NIR-mbc94, a Fluorescent Ligand that Binds to Endogenous CB2 Receptors and Is Amenable to High-Throughput Screening

Abstract

SUMMARY High-throughput screening (HTS) of chemical libraries is often used for the unbiased identification of compounds interacting with G protein-coupled receptors (GPCRs), the largest family of therapeutic targets. However, current HTS methods require removing GPCRs from their native environment, which modifies their pharmacodynamic properties and biases the screen toward false positive hits. Here, we developed and validated a molecular imaging (MI) agent, NIR-mbc94, which emits near infrared (NIR) light and selectively binds to endogenously expressed cannabinoid CB2 receptors, a recognized target for treating autoimmune diseases, chronic pain and cancer. The precision and ease of this assay allows for the HTS of compounds interacting with CB2 receptors expressed in their native environment.

In Vivo Type 2 Cannabinoid Receptor-Targeted Tumor Optical Imaging Using a Near Infrared Fluorescent Probe

“…In addition, CB 2 R-targeted imaging could help to elucidate the exact role of CB 2 R in cancer progression as well as its potential as a therapeutic target and could be applied to cancer diagnosis, therapeutic monitoring, and high-throughput drug screening. 20 …”

“…These results indicate specific binding of NIR760-mbc94 to the target receptor and are consistent with our previous cellular imaging studies using another fluorescent CB 2 R probe. 8,20 , As a more quantitative approach, a multiplate reader system was used to analyze cellular uptake of NIR760-mbc94 (Figure 5b). Cells treated with NIR760-mbc94 showed a 4-fold higher fluorescence signal than those with free NIR 760 dye ( p < 0.001), and blocking with SR144528 (10 µM) resulted in an approximately 40% decrease in fluorescence intensity for cells with NIR760-mbc94 ( p < 0.01).…”

A novel near-infrared fluorescence imaging probe that preferentially binds to cannabinoid receptors CB2R over CB1R

“…[14] However, it is difficult to develop CB 2 R-targeted fluorescent probes because conjugating a relatively large fluorescent dye to a CB 2 R ligand could obliterate its binding to CB 2 R.[17] In fact, even attaching a small linker to a CB 2 R ligand often leads to the loss of binding. [18] We recently overcame the difficulty and introduced NIR dyes to strategic positions of a pyrazole and quinolone structure respectively. [19-23] The specificity of these probes was demonstrated by blocking studies both in vitro and in vivo.…”

Targeted zwitterionic near infrared fluorescent probe for improved imaging of type 2 cannabinoid receptors

“…2 In a number of additional cell types under disease conditions, CB 2 R expression is greatly up-regulated. 3 The richness of CB 2 R involvement has positioned the receptor as an attractive therapeutic target for treating an array of pathologies, such as cancers, 4,5 neurodegenerative diseases, 2,6 inflammation, 7,8 pain, 9 osteoporosis, 10 immunological disorders, [11][12][13] and drug abuse. 14 As such, there has been an explosion of research over the past 12 years that focused on the biology and therapeutic promises of CB 2 R. However, the precise role of CB 2 R in the regulation of diseases remains unclear.…”

“…21 CB 2 R expression also increases in diseased central nervous system cells, including astrocytomas, 22 activated microglia and neurons, 14,15,23 astrocytes of Alzheimer's disease, 24 as well as T-lymphocytes, microglia, and astrocytes in multiple sclerosis. 25 Because CB 2 R expression is nearly absent in these areas under basal conditions, and increases as much as 100-fold in diseases, 3 CB 2 R imaging can achieve a high-imaging contrast in diseased tissues. Accordingly, CB 2 R is a promising target for MI applications.…”