The Cannabinoid 2 receptor, CB2R, belonging to the endocannabinoid system, ECS, is involved in the first steps of neurodegeneration and cancer evolution and progression and thus its modulation may be exploited in the therapeutic and diagnostic fields. However, CB2Rs distribution and signaling pathways in physiological and pathological conditions are still controversial mainly because of the lack of reliable diagnostic tools. With the aim to produce green and safe systems to detect CB2R, we designed a series of fluorescent ligands with three different green fluorescent moieties (4-dimethylaminophthalimide, 4-DMAP, 7-nitro-4-yl-aminobenzoxadiazole, NBD, and Fluorescein-thiourea, FTU) linked to the N1-position of the CB2R pharmacophore N-adamantyl-4-oxo-1,4-dihydroquinoline-3-carboxamide through polymethylene chains. Compound 28 emerged for its compromise between good pharmacodynamic properties (CB2R Ki = 130 nM and no affinity vs the other subtype CB1R) and optimal fluorescent spectroscopic properties. Therefore, compound 28 was studied through FACS (saturation and competitive binding studies) and fluorescence microscopy (visualization and competitive binding) in engineered cells (CB2R-HEK293 cells) and in diverse tumour cells. The fluoligand binding assays were successfully set up, and affinity values for the two reference compounds GW405833 and WIN55,212-2, comparable to the values obtained by radioligand binding assays, were obtained. Fluoligand 28 also allowed the detection of the presence and quantification of the CB2R in the same cell lines. The interactions of compound 28 within the CB2R binding site were also investigated by molecular docking simulations, and indications for the improvement of the CB2R affinity of this class of compounds were provided. Overall, the results obtained through these studies propose compound 28 as a safe and green alternative to the commonly used radioligands for in vitro investigations.

Design and synthesis of fluorescent ligands for the detection of cannabinoid type 2 receptor (CB2R)

Riganti C.;Kopecka J.;Infantino V.;Abate C.;
2020

Abstract

The Cannabinoid 2 receptor, CB2R, belonging to the endocannabinoid system, ECS, is involved in the first steps of neurodegeneration and cancer evolution and progression and thus its modulation may be exploited in the therapeutic and diagnostic fields. However, CB2Rs distribution and signaling pathways in physiological and pathological conditions are still controversial mainly because of the lack of reliable diagnostic tools. With the aim to produce green and safe systems to detect CB2R, we designed a series of fluorescent ligands with three different green fluorescent moieties (4-dimethylaminophthalimide, 4-DMAP, 7-nitro-4-yl-aminobenzoxadiazole, NBD, and Fluorescein-thiourea, FTU) linked to the N1-position of the CB2R pharmacophore N-adamantyl-4-oxo-1,4-dihydroquinoline-3-carboxamide through polymethylene chains. Compound 28 emerged for its compromise between good pharmacodynamic properties (CB2R Ki = 130 nM and no affinity vs the other subtype CB1R) and optimal fluorescent spectroscopic properties. Therefore, compound 28 was studied through FACS (saturation and competitive binding studies) and fluorescence microscopy (visualization and competitive binding) in engineered cells (CB2R-HEK293 cells) and in diverse tumour cells. The fluoligand binding assays were successfully set up, and affinity values for the two reference compounds GW405833 and WIN55,212-2, comparable to the values obtained by radioligand binding assays, were obtained. Fluoligand 28 also allowed the detection of the presence and quantification of the CB2R in the same cell lines. The interactions of compound 28 within the CB2R binding site were also investigated by molecular docking simulations, and indications for the improvement of the CB2R affinity of this class of compounds were provided. Overall, the results obtained through these studies propose compound 28 as a safe and green alternative to the commonly used radioligands for in vitro investigations.
188
112037
112056
Cannabinoid receptors; CB2R; CB2R fluorescent ligands; Flow cytometry study; Fluorescent competitive binding assay; Fluorescent probes
Spinelli F.; Giampietro R.; Stefanachi A.; Riganti C.; Kopecka J.; Abatematteo F.S.; Leonetti F.; Colabufo N.A.; Mangiatordi G.F.; Nicolotti O.; Perrone M.G.; Brea J.; Loza M.I.; Infantino V.; Abate C.; Contino M.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2318/1747150
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