Design and development of probes for studying cannabinoid and related orphan G protein-coupled receptors and their signaling

Abstract

G protein-coupled receptors (GPCRs) are the largest family of human membrane proteins and represent one of the most important classes of pharmacological targets for drugs. Among these, the cannabinoid (CB)receptor subtypes CB₁ and CB₂ have emerged as attractive targets due to their roles in various pathophysiological processes. Growing evidence indicates that cannabinoids can additionally interact with other GPCRs beyond CB₁ and CB₂ receptors, in particular with the orphan receptors GPR18 and GPR55. Both receptors have potential as drug targets, but conflicting findings have presented significant challenges in their validation for therapeutic application. <br /> The present study aimed to design, synthesize, evaluate, and characterize tool compounds, for GPR18, CB₁ and CB₂ receptors. Through screening campaigns, structure–activity relationship (SAR) analysis, and broad pharmacological evaluation, several potent and reliable tools were developed. <br /> For GPR18, the initial hit compounds <strong>PSB-KD107</strong> (EC₅₀ = 0.562 µM) and <strong>PSB-KD477</strong> (EC₅₀ = 0.454 µM) were used as starting points to yield more potent and selective agonists, including <strong>PSB-KK1415</strong> (EC₅₀ = 0.0191 µM), <strong>PSB-KK1445</strong> (EC₅₀ = 0.0454 µM), and <strong>PSB-KK1418</strong> (EC₅₀ = 0.0711 µM). These compounds present the most potent (<strong>PSB-KK1415</strong>), most selective (<strong>PSB-KK1445</strong>)<strong> </strong>and most efficacious (<strong>PSB-KK1418</strong>) GPR18 agonists known to date. Additionally, <strong>PSB-KK1846</strong> was identified as the most potent antagonist for GPR55, with an IC₅₀ of 0.884 µM. The new agonists were found to display minimal species differences. In contrast, &Delta;⁹-tetrahydrocannabinol (THC) – a standard tool compound for this receptor, acted as a weak partial agonist at the mouse GPR18 receptor, and therefore cannot be used for mouse studies of GPR18. <br /> For CB₂ receptors, a fluorinated indole derivative, demonstrated high selectivity over other CB and CB-like receptors and exhibited a favorable profile in metabolic stability assays, making it a promising candidate for radiotracer development for positron emission tomography (PET). Diindolylmethane (DIM) and its derivatives were optimized to generate tool compounds for CB₂ receptors with distinct pharmacological properties. <strong>PSB-19837</strong> (EC₅₀ = 0.0144 µM) was identified as the most potent CB₂ agonist in the present series. <strong>PSB-19571</strong> exhibited biased agonism toward β-arrestin (6-fold preference for β-arrestin-2 over G-protein signaling), while <strong>PSB-18691</strong> showed a Ga_i-protein bias (20-fold preference for G-protein over β-arrestin-2 signaling). <br /> A further study focused on the adenosine A_2A receptor, presenting the crystal structure of the receptor bound to the partial agonist LUF5834, with an atomic resolution of 2.3 Å. Structural studies revealed previously unobserved binding interactions, including the interaction of the phenolic group of LUF5834 with transmembrane helix III, and an ionic lock between the extracellular loops. Pharmacological studies confirmed that LUF5834 acts as a partial agonist, likely stabilizing an equilibrium between active and inactive receptor states. <br /> In conclusion, the present work identified, optimized and broadly characterized small molecules that serve as potent tool compounds for GPR18 and CB₂receptors. In addition, structural and pharmacological studies on the adenosine A_2A receptor provided valuable insights into the mechanism of partial agonism at this prototypic GPCR.