A Novel Selective Inverse Agonist of the CB₂ Receptor as a Radiolabeled Tool Compound for Kinetic Binding Studies

Abstract

The endocannabinoid system, and in particular the cannabinoid type 2 receptor (CB2R), raised the interest of many medicinal chemistry programs for its therapeutic relevance in several (patho)physiologic processes. However, the physico-chemical properties of tool compounds for CB2R (e.g., the radioligand [H]CP55,940) are not optimal, despite the research efforts in developing effective drugs to target this system. At the same time, the importance of drug-target binding kinetics is growing since the kinetic binding profile of a ligand may provide important insights for the resulting in vivo efficacy. In this context we synthesized and characterized [H]RO6957022, a highly selective CB2R inverse agonist, as a radiolabeled tool compound. In equilibrium and kinetic binding experiments [H]RO6957022 showed high affinity for human CB2R with fast association () and moderate dissociation () kinetics. To demonstrate the robustness of [H]RO6957022 binding, affinity studies were carried out for a wide range of CB2R reference ligands, spanning the range of full, partial, and inverse agonists. Finally, we used [H]RO6957022 to study the kinetic binding profiles (i.e., and values) of selected synthetic and endogenous (i.e., 2-arachidonoylglycerol, anandamide, and noladin ether) CB2R ligands by competition association experiments. All tested ligands, and in particular the endocannabinoids, displayed distinct kinetic profiles, shedding more light on their mechanism of action and the importance of association rates in the determination of CB2R affinity. Altogether, this study shows that the use of a novel tool compound, i.e., [H]RO6957022, can support the development of novel ligands with a repertoire of kinetic binding profiles for CB2R.

Development of High-Specificity Fluorescent Probes to Enable Cannabinoid Type 2 Receptor Studies in Living Cells

“…The determined kinetic rate constants are in good agreement with literature data derived using a CB2R selective radioligand. 51 As compared to HU-308, the higher affinity of SR144528 to hCB2R is driven by a faster on-rate, with otherwise equivalent off-rates.…”

“…Radioligands are ubiquitously used to study GPCR expression as well as ligand binding affinities and kinetics. 51 Safety concerns, radioactive waste management, and, in the case of filtration assays, the need for iterative washing steps limit the applicability of radioligand-based assays for high throughput screening (HTS). Fluorescent probes are devoid of such drawbacks and thus, in conjunction with FRET-based assays, offer an attractive solution towards the development of high throughput equilibrium binding assays.…”

Investigating the Influence of Tracer Kinetics on Competition-Kinetic Association Binding Assays: Identifying the Optimal Conditions for Assessing the Kinetics of Low-Affinity Compounds

“…State of the art radioligand-based competition association binding assays are routinely formulated using only 12 time points (Sykes et al, 2010) employing either a single concentration of competitor (Sykes et al, 2014;Martella et al, 2017) or up to three concentrations of competitor (Sykes et al, 2009(Sykes et al, , 2010) and a tracer concentration in the range of 1-10Â its own K d . Current knowledge of FRET-based competition association binding assays is based on a small number of studies, which in general have employed an off-line addition protocol to improve experimental throughput but also to allow greater temperature control during the initial mixing step (Klein-Herenbrink et al, 2016;Sykes et al, 2017;.…”

Development of High-Specificity Fluorescent Probes to Enable Cannabinoid Type 2 Receptor Studies in Living Cells

“…The determined kinetic rate constants are in good agreement with literature data derived using a CB 2 R-selective radioligand. 54 As compared to HU-308, the higher affinity of SR144528 to hCB 2 R is driven by a faster on-rate, with otherwise equivalent off-rates.…”

“…Radioligands are ubiquitously used to study GPCR expression as well as ligand binding affinities and kinetics. 54 Safety concerns, radioactive waste management, and, in the case of filtration assays, the need for iterative washing steps limit the applicability of radioligand-based assays for high-throughput screening (HTS). Fluorescent probes are devoid of such drawbacks and thus, in conjunction with FRET-based assays, offer attractive solutions toward the development of highthroughput equilibrium binding assays.…”