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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 CB1 orthosteric 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.

SUBMITTER: Bruno A

PROVIDER: S-EPMC3895875 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Beyond radio-displacement techniques for identification of CB1 ligands: the first application of a fluorescence-quenching assay.

Bruno Agostino A Lembo Francesca F Novellino Ettore E Stornaiuolo Mariano M Marinelli Luciana L

Scientific reports 20140120

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 CB1 orthosteric 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 ...[more]

PMID: 24441508

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Project description:The regulator of expression of virion (Rev) protein binds specifically to the Rev-responsive element (RRE) RNA in order to regulate the expression of the human immunodeficiency virus (HIV)-1 genes. Fluorescence indicator displacement assays have been used to identify ligands that can inhibit the Rev–RRE interaction; however, the small fluorescence indicators cannot fully replace the Rev peptide or protein. As a result, a single rhodamine B labeled Rev (RB-Rev) model peptide was utilized in this study to develop a direct and efficient Rev–RRE inhibitor screening model. Due to photon-induced electron transfer quenching of the tryptophan residue on the RB fluorophore, the fluorescence of RB in Rev was weakened and could be dramatically reactivated by interaction with RRE RNA in ammonium acetate buffer (approximately six times). The interaction could reduce the electron transfer between tryptophan and RB, and RRE could also increase RB fluorescence. The inhibitor screening model was evaluated using three known positive Rev–RRE inhibitors, namely, proflavin, 6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine (ICR 191), and neomycin, as well as a negative drug, arginine. With the addition of the positive drugs, the fluorescence of the Rev–RRE decreased, indicating the displacement of RB-Rev. This was confirmed using atomic force microscopy (AFM) and the fluorescence was essentially unaffected by the addition of arginine. The results demonstrated that RB-Rev can be used as a fluorescent probe for recognizing small ligands that target RRE RNA. The Rev–RRE inhibitor screening model offers a novel approach to evaluating and identifying long-acting Rev inhibitors. Graphical abstract Image 1 Highlights • Fluorescence enhancement of RB-Rev after interaction with RRE was discovered.• RB-Rev was used as a fluorescent probe for recognizing small ligands that target RRE RNA.• A direct fluorescence “turn on” Rev–RRE inhibitor screening model was developed.

Dataset Information

Beyond radio-displacement techniques for identification of CB1 ligands: the first application of a fluorescence-quenching assay.

Publications

Beyond radio-displacement techniques for identification of CB1 ligands: the first application of a fluorescence-quenching assay.

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