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Cannabinoid 1 Receptor Mutants and Covalent Cannabinergic Probes for Ligand Binding Site Characterization
Author(s) -
Korde Anisha,
Zvonok Nikolai,
Vemuri Kiran,
Makriyannis Alexandros
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.770.12
Subject(s) - chemistry , cannabinoid , radioligand , cannabinoid receptor , binding site , receptor , ligand (biochemistry) , cysteine , biochemistry , stereochemistry , agonist , enzyme
Cannabinoid receptor 1 (CB1) modulation using xenobiotic or endogenous ligands has potential in numerous medical conditions including treatment of obesity, osteoporosis, pain, inflammation, cancer, neurodegenerative disorders, drug abuse, and cardio metabolic diseases. Due to the lack of the CB1 structural data, characterization of the receptor binding sites using covalent probes and CB1 mutants is an essential tool for design of high affinity, selective ligands. HEK293F cell lines stably expressing either the wild type (WT) or selected single or double cysteine hCB1 receptor mutants were generated. A radioligand‐binding assay identified a significantly higher density (3‐5 fold) of the hCB1 receptor in HEK293F membrane preparations, than in any of our previously used mammalian cell lines. Binding affinities to the hCB1 receptor mutants at the orthosteric‐binding site were evaluated in radioligand binding assays using [ 3 H] CP55940, [ 3 H] WIN‐55212‐2 and [ 3 H] SR‐141716‐A. The new hCB1 HEK293F cell lines were utilized for obtaining experimental information on the binding site of covalent biarylpyrazole derivatives of the CB1‐selective antagonist SR141716A. The SR141716A analogue with electrophilic isothiocyanate group covalently attached exclusively to the cysteine at position 386 (7.42) in the transmembrane helix 7 (THM7) of hCB1. Other SR141716A analogues, containing nitrate or mesylate groups targeted the cysteine at position 382 (7.38) in the THM7 of hCB1. Probably the difference in chain length of the attached chemotype is responsible for discrimination between cysteine 382 and 386. These results provide important direct information in modeling of SR141716A binding to CB1 receptor.
NIH/NIDA Grants D3801, DA7215&DA9158.