Patent review of cannabinoid receptor type 2 (CB2R) modulators (2016-present).

INTRODUCTION: Cannabinoid receptor type 2 (CB2R), predominantly expressed in immune tissues, is believed to play a crucial role within the body's protective mechanisms. Its modulation holds immense therapeutic promise for addressing a wide spectrum of dysbiotic conditions, including cardiovascular, gastrointestinal, liver, kidney, neurodegenerative, psychiatric, bone, skin, and autoimmune diseases, as well as lung disorders, cancer, and pain management. AREAS COVERED: This review is an account of patents from 2016 up to 2023 which describes novel CB2R ligands, therapeutic applications, synthesis, as well as formulations of CB2R modulators. EXPERT OPINION: The patents cover a vast, structurally diverse chemical space. The focus of CB2R ligand development has shifted from unselective dual-cannabinoid receptor type 1 (CB1R) and 2 agonists toward agonists with high selectivity over CB1R, particularly for indications associated with inflammation and tissue injury. Currently, there are at least eight CB2R agonists and one antagonist in active clinical development. A better understanding of the endocannabinoid system (ECS) and in particular of CB2R pharmacology is required to unlock the receptor's full therapeutic potential.

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

We report a blueprint for the rational design of G protein coupled receptor (GPCR) ligands with a tailored functional response. The present study discloses the structure-based design of cannabinoid receptor type 2 (CB2R) selective inverse agonists (S)-1 and (R)-1, which were derived from privileged agonist HU-308 by introduction of a phenyl group at the gem-dimethylheptyl side chain. Epimer (R)-1 exhibits high affinity for CB2R with Kd = 39.1 nM and serves as a platform for the synthesis of a wide variety of probes. Notably, for the first time these fluorescent probes retain their inverse agonist functionality, high affinity, and selectivity for CB2R independent of linker and fluorophore substitution. Ligands (S)-1, (R)-1, and their derivatives act as inverse agonists in CB2R-mediated cAMP as well as G protein recruitment assays and do not trigger ß-arrestin-receptor association. Furthermore, no receptor activation was detected in live cell ERK1/2 phosphorylation and Ca2+-release assays. Confocal fluorescence imaging experiments with (R)-7 (Alexa488) and (R)-9 (Alexa647) probes employing BV-2 microglial cells visualized CB2R expressed at endogenous levels. Finally, molecular dynamics simulations corroborate the initial docking data in which inverse agonists restrict movement of toggle switch Trp2586.48 and thereby stabilize CB2R in its inactive state.

Platform Reagents Enable Synthesis of Ligand-Directed Covalent Probes: Study of Cannabinoid Receptor 2 in Live Cells.

Pharmacological modulation of cannabinoid receptor type 2 (CB2R) holds promise for the treatment of neuroinflammatory disorders, such as Alzheimer's disease. Despite the importance of CB2R, its expression and downstream signaling are insufficiently understood in disease- and tissue-specific contexts. Herein, we report the first ligand-directed covalent (LDC) labeling of CB2R enabled by a novel synthetic strategy and application of platform reagents. The LDC modification allows visualization and study of CB2R while maintaining its ability to bind other ligands at the orthosteric site. We employed in silico docking and molecular dynamics simulations to guide probe design and assess the feasibility of LDC labeling of CB2R. We demonstrate selective, covalent labeling of a peripheral lysine residue of CB2R by exploiting fluorogenic O-nitrobenzoxadiazole (O-NBD)-functionalized probes in a TR-FRET assay. The rapid proof-of-concept validation with O-NBD probes inspired incorporation of advanced electrophiles suitable for experiments in live cells. To this end, novel synthetic strategies toward N-sulfonyl pyridone (N-SP) and N-acyl-N-alkyl sulfonamide (NASA) LDC probes were developed, which allowed covalent delivery of fluorophores suitable for cellular studies. The LDC probes were characterized by a radioligand binding assay and TR-FRET experiments. Additionally, the probes were applied to specifically visualize CB2R in conventional and imaging flow cytometry as well as in confocal fluorescence microscopy using overexpressing and endogenously expressing microglial live cells.

Reverse Design toward Optimized Labeled Chemical Probes - Examples from the Endocannabinoid System.

Labeled chemical probes are of utmost importance to bring drugs from the laboratory through the clinic and ultimately to market. They support and impact all research and discovery phases: target verification and validation; assay development; lead optimization; and biomarker engagement in the context of preclinical studies and human trials. Probes should display high potency and selectivity as well as fulfill specific criteria in connection with absorption, distribution, metabolism, excretion and toxicology (ADMET) profile. Progress in fields such as imaging and proteomics increased the need for specialized probes to support drug discovery. Labeled probes carrying an additional reporter group are valuable tools to meet specific application requirements, but pose significant challenges in design and construction. In the reverse-design approach, small molecules previously optimized in medicinal chemistry programs form the basis for the generation of such high-quality probes. We discuss the reverse design concept for the generation of labeled probes targeting the endocannabinoid system (ECS), a complex lipid signaling network that plays a key role in many human health and disease conditions. The examples highlighted include diverse reporter units for a range of applications. In several cases the reported probes were the product of mutually rewarding and highly cross-fertilizing collaborations among academic and industry research programs, a strategy that can serve as a blueprint for future probe generation efforts.

A Catch-and-Release Approach to Selective Modification of Accessible Tyrosine Residues.

The tyrosine side chain is amphiphilic leading to significant variations in the surface exposure of tyrosine residues in the folded structure of a native sequence protein. This variability can be exploited to give residue-selective functionalization of a protein substrate by using a highly reactive diazonium group tethered to an agarose-based resin. This novel catch-and-release approach to protein modification has been demonstrated for proteins with accessible tyrosine residues, which are compared with a control group of proteins in which there are no accessible tyrosine residues. MS analysis of the modified proteins showed that functionalization was highly selective, but reactivity was further attenuated by the electrostatic environment of any individual residue. Automated screening of PDB structures allows identification of potential candidates for selective modification by comparison with the accessibility of the tyrosine residue in a benchmark peptide (GYG).