Beyond radio-displacement techniques for Identification of CB1 Ligands: The First Application of a Fluorescence-quenching Assay

Abstract

Cannabinoid type 1 Receptor (CB1) belongs to the GPCR family and it has been targeted, so far, for the discovery of drugs aimed at the treatment of neuropathic pain, nausea, vomit, and food intake disorders. Here, we present the development of the first fluorescent assay enabling the measurement of kinetic binding constants for CB1orthosteric ligands. The assay is based on the use of T1117, a fluorescent analogue of AM251. We prove that T1117 binds endogenous and recombinant CB1 receptors with nanomolar affinity. Moreover, T1117 binding to CB1 is sensitive to the allosteric ligand ORG27569 and thus it is applicable to the discovery of new allosteric drugs. The herein presented assay constitutes a sustainable valid alternative to the expensive and environmental impacting radiodisplacement techniques and paves the way for an easy, fast and cheap high-throughput drug screening toward CB1 for identification of new orthosteric and allosteric modulators.

Synthesis, biological activity and molecular modeling of new biphenylic carboxamides as potent and selective CB2 receptor ligands

“…To rationalize SARs displayed by compounds 4e12 (Table 1), three-dimensional models of both CB1 and CB2 receptors were generated, as previously reported by us [42], by means of Modeller 9.11 software. Basically, the sphingoshine-1-phosphate type 1 receptor [S1P 1 (PDB code: 3V2Y)] was used as template because of both, the sequence identity percentage (27.03% and 24.16% for CB1 and CB2, respectively) and the close evolutionary relationship [43] (see Material and Methods).…”

“…Three-dimensional models of both CB1 and CB2 receptors were generated through the Modeller 9.11 software [49] as previously reported by us [42]. Basically, the S1P 1 X-ray crystal structure (pdb code: 3V2Y [50]) was chosen and used as template to generate the 3D structure of both CB1 and CB2 receptors.…”

A potent fluorescent calcitonin gene‐related peptide analogue enables visualization of receptor internalization

“…Fluorescent‐based techniques, such as the development of fluorescently labeled ligands, are useful for analyzing peptide‐receptor interactions and binding kinetics . Furthermore they can help to elucidate the mechanisms of receptor trafficking and internalization, and could be used in the development of competition binding assays as a safer alternative to radioactive ligands . Despite their potential, few potent fluorescent ligands for class B GPCRs exist.…”

Chemical Tools for Studying Lipid-Binding Class A G Protein–Coupled Receptors

“…T1117 was then used as the competitive tracer in a binding assay, providing IC 50 values for anandamide and AM251 in agreement with literature values. Bruno et al (2014) further demonstrated utility of T1117 as a drug discovery tool by the appropriate identification of the CB 1 receptor allosteric ligand ORG27569 (Fig. 6).…”