Flipping the GPCR Switch: Structure-Based Development of Selective Cannabinoid Receptor 2 Inverse Agonists.

Area of Science:

• Medicinal Chemistry
• Pharmacology
• Molecular Biology

Background:

• G protein-coupled receptors (GPCRs) are crucial drug targets.
• Cannabinoid receptor type 2 (CB2R) is implicated in various physiological processes.
• Developing selective CB2R ligands with defined functional outcomes is challenging.

Purpose of the Study:

• To rationally design novel CB2R selective inverse agonists.
• To create a versatile platform for synthesizing functional fluorescent CB2R probes.
• To investigate the molecular mechanism underlying CB2R inverse agonism.

Main Methods:

• Structure-based drug design and synthesis of novel ligands ((S)-1 and (R)-1).
• Biochemical assays measuring CB2R inverse agonism (cAMP, G protein recruitment, β-arrestin assays).
• Cell-based assays (ERK1/2 phosphorylation, Ca2+ release) and live-cell confocal fluorescence imaging.
• Molecular dynamics simulations.

Main Results:

• Developed CB2R selective inverse agonists (S)-1 and (R)-1 with high affinity (Kd = 39.1 nM for (R)-1).
• Synthesized novel fluorescent CB2R probes retaining inverse agonism, affinity, and selectivity.
• Demonstrated inverse agonism without triggering β-arrestin, ERK1/2 phosphorylation, or Ca2+ release.
• Visualized endogenous CB2R in microglial cells using fluorescent probes.
• Molecular dynamics simulations confirmed stabilization of the CB2R inactive state.

Conclusions:

• A successful blueprint for designing tailored GPCR functional ligands.
• Established a novel class of CB2R inverse agonists and versatile fluorescent probes.
• Provided mechanistic insights into CB2R inverse agonism via stabilization of the inactive receptor state.