Cannabinoid type 2 receptor (CB 2 R) is an attractive target for the treatment of pain and inflammatory disorders. Availability of a selective CB 2 R fluorescent ligand to study CB 2 R expression and localization in healthy and disease conditions would greatly contribute to improving our understanding of this receptor. Herein, we report a series of chromenopyrazole-based CB 2 R fluorescent ligands. The highest affinity fluorescent ligand was Cy5-containing 24 (hCB 2 R pK i = 7.38 ± 0.05), which had 131-fold selectivity over CB 1 R. In a cAMP BRET assay, 24 behaved as a potent CB 2 R inverse agonist. Widefield imaging experiments showed that 24 binds to CB 2 R in live cells with good selectivity and low levels of nonspecific fluorescence. The high affinity, selectivity, and suitable imaging properties of fluorescent ligand 24 make it a valuable tool for studying CB 2 R.

High affinity, cannabinoid type 2 receptor selective ligand.

Cannabinoid, free fatty acid, lysophosphatidic acid, sphingosine 1-phosphate, prostanoid, leukotriene, bile acid, and platelet-activating factor receptor families are class A G protein-coupled receptors with endogenous lipid ligands. Pharmacological tools are crucial for studying these receptors and addressing the many unanswered questions surrounding expression of these receptors in normal and diseased tissues. An inherent challenge for developing tools for these lipid receptors is balancing the often lipophilic requirements of the receptor-binding pharmacophore with favorable physicochemical properties to optimize highly specific binding. In this study, we review the radioligands, fluorescent ligands, covalent ligands, and antibodies that have been used to study these lipid-binding receptors. For each tool type, the characteristics and design rationale along with in vitro and in vivo applications are detailed.

In this review, we highlight bifunctional modalities that perform functions other than degradation and have great potential to revolutionize disease treatment, while also serving as important tools in basic research to explore new aspects of biology.

Chimeric small molecules that induce post-translational modification (PTM) on a target protein by bringing it in proximity to a PTM-inducing enzyme are furnishing novel modalities to perturb protein function. Despite recent advances, such molecules are unavailable for a critical PTM, tyrosine phosphorylation. Furthermore, the contemporary design paradigm of chimeric molecules, formed by joining a non-inhibitory binder of the PTM-inducing enzyme with the binder of the target protein, prohibits the recruitment of most PTM-inducing enzymes as their non-inhibitory binders are unavailable. Here, we report two platforms to generate phosphorylation-inducing chimeric small molecules (PHICS) for tyrosine phosphorylation. We generate PHICS from both non-inhibitory binders (scantily available, platform 1) and kinase inhibitors (abundantly available, platform 2) using cysteine-based group transfer chemistry. PHICS triggered phosphorylation on tyrosine residues in diverse sequence contexts and target proteins (e.g., membrane-associated, cytosolic) and displayed multiple bioactivities, including initiation of a growth receptor signaling cascade and death of drug-resistant cancer cells. These studies provide an approach to induce biologically relevant PTM and lay the foundation for pharmacologic PTM editing (i.e., induction or removal) on target proteins using abundantly available inhibitors of PTM-inducing or erasing enzymes.TOC