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

Abstract

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.

Rational Design, Synthesis, and Evaluation of Fluorescent CB2 Receptor Ligands for Live-Cell Imaging: A Comprehensive Review

“…The signal seen demonstrated colocalization between the SNAP-dye signal in the far-red range and the signal of interest. This colocalization provided confirmation that the tagging of SNAP tagged CB 2 Rs was effective using compound 29 [ 79 ].…”

Optochemical profiling of receptor-proximal proteins in vivo in minutes

“…Ligand-directed chemical reagents have been developed by multiple groups for various receptors over the years as proof of its generality as a photosensitizer anchoring strategy. 49,50 We have additionally con rmed that nanobody-photosensitizer conjugates, accessible via a facile one-step reaction, can also be used for in vivo PhoxID (Extended Data Fig. 8 and Supplementary Table 6).…”

Lysine-Reactive N-Acyl-N-aryl Sulfonamide Warheads: Improved Reaction Properties and Application in the Covalent Inhibition of an Ibrutinib-Resistant BTK Mutant

“…It is also worth mentioning that the ArNASA warheads could be used as cleavable electrophiles for ligand-directed covalent protein labeling, which is the unique advantage of ArNASA over the other cysteine- and lysine-targeting warheads. , Given the recent successful studies on NASA chemistry, not only by us, but also by other groups, − , the excellent reactivity and biocompatibility of ArNASA warheads may be leveraged to further expand the breadth of applications for protein activity control and engineering techniques using covalent modification.…”

“…17,18 Although ∼99.9% of the ε-amino groups are theoretically protonated at physiological pH (7.4) and thereby exhibit low reactivity, several lysine-targetable warheads, including activated esters, 19 sulfonyl fluorides, 20−23 fluorosulfates, 24 vinyl sulfones, 25 and aldehydes, 26−29 in the development of a noncatalytic lysine-targeting warhead. 30 The excellent reactivity of NASA toward lysine has enabled a variety of applications, including prolonged inhibition of intracellular proteins, 30,31 an aptamer-based TCI, 32 ligand-directed protein labeling, 30,[33][34][35][36][37]52 and proteome-wide ligandable lysine profiling. 38 However, NASAbased reagents often suffer from rapid inactivation by hydrolysis and off-target reactions in protein-rich environments, such as serum-containing media that is essential for cell culture, because of the relatively high intrinsic reactivity.…”

“…Our group has recently also found that N -acyl- N -alkyl sulfonamides (NASAs) have great potential in the development of a noncatalytic lysine-targeting warhead . The excellent reactivity of NASA toward lysine has enabled a variety of applications, including prolonged inhibition of intracellular proteins, , an aptamer-based TCI, ligand-directed protein labeling, ,− , and proteome-wide ligandable lysine profiling . However, NASA-based reagents often suffer from rapid inactivation by hydrolysis and off-target reactions in protein-rich environments, such as serum-containing media that is essential for cell culture, because of the relatively high intrinsic reactivity.…”