Deciphering complexity of GPCR signaling and modulation: implications and perspectives for drug discovery.

G protein-coupled receptors (GPCRs) are central to pathophysiological processes and remain prominent targets in drug discovery. Recent advances in understanding GPCR signaling and modulation, such as biased agonism, dual agonism, and non-canonical G protein signaling, have expanded the therapeutic landscape of these receptors. These understandings have led (and are leading further) to innovative approaches that broaden GPCRs as therapeutic targets, going after better efficacy and minimizing adverse effects. However, tachyphylaxis, a rapid decrease in receptor responsiveness after repeated stimulation, presents a significant challenge in a chronic treatment context. Recent findings from our group revealed that tachyphylaxis in the angiotensin type 1 (AT1) receptor is primarily governed by the ligand's dissociation rate (koff), i.e. high residence time, rather than by β-arrestin-mediated desensitization, as could be expected. This suggests that internalized AT1 receptors remain active when bound to ligands with high residence time, favoring sustained signaling from endosomes. Importantly, the concept of high residence time sheds new light on intracellular signaling phenomena and underscores the therapeutic value of modulating intracellular receptor activity, including the development of novel cell-permeant antagonists. This review discusses critical pharmacological parameters for drug discovery focused on agonists, including (i) activation of preferential signaling pathways (biased agonism), (ii) internalization/recycling rates, (iii) tachyphylaxis/desensitization, (iv) allosteric modulators, and (v) intracellular receptor signaling and its blockade, emphasizing the need for strategies that extend beyond conventional GPCRs' functional assays. Additionally, this review highlights how advancements in high-resolution imaging, bioluminescence resonance energy transfer-based biosensors, and computational modeling are crucial for elucidating complex GPCRs' behaviors, particularly in understanding mechanisms like tachyphylaxis and its interplay with compartment-specific signaling. These approaches not only pave the way for therapies that strategically leverage or mitigate tachyphylaxis to sustain receptor responsiveness, but could enable the design of drugs targeting intracellular pathways as a strategy to enhance efficacy and minimize adverse effects. These insights underscore the importance of integrating diverse pharmacological strategies to refine GPCR-targeted therapies and address unmet medical needs, particularly in chronic conditions where sustained receptor activity is critical.