Beyond radio-displacement techniques for Identification of CB(1) Ligands: The First Application of a Fluorescence-quenching Assay

Cannabinoid type 1 Receptor (CB(1)) 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 CB(1)orthosteric ligands. The assay is based on the use of T1117, a fluorescent analogue of AM251. We prove that T1117 binds endogenous and recombinant CB(1) receptors with nanomolar affinity. Moreover, T1117 binding to CB(1) 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 CB(1) for identification of new orthosteric and allosteric modulators.