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Identification of a novel “almost neutral” μ‐opioid receptor antagonist in CHO cells expressing the cloned human μ‐opioid receptor
Author(s) -
Sally Elliott J.,
Xu Heng,
Dersch Christina M.,
Hsin LingWei,
Chang LiTe,
Prisinzano Thomas E.,
Simpson Denise S.,
Giuvelis Denise,
Rice Kenner C.,
Jacobson Arthur E.,
Cheng Kejun,
Bilsky Edward J.,
Rothman Richard B.
Publication year - 2010
Publication title -
synapse
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.809
H-Index - 106
eISSN - 1098-2396
pISSN - 0887-4476
DOI - 10.1002/syn.20723
Subject(s) - inverse agonist , agonist , receptor , chemistry , partial agonist , opioid receptor , chinese hamster ovary cell , μ opioid receptor , pharmacology , stereochemistry , endocrinology , medicine , microbiology and biotechnology , biochemistry , biology
The basal (constitutive) activity of G protein‐coupled receptors allows for the measurement of inverse agonist activity. Some competitive antagonists turn into inverse agonists under conditions where receptors are constitutively active. In contrast, neutral antagonists have no inverse agonist activity, and they block both agonist and inverse agonist activity. The μ‐opioid receptor (MOR) demonstrates detectable constitutive activity only after a state of dependence is produced by chronic treatment with a MOR agonist. We therefore sought to identify novel MOR inverse agonists and novel neutral MOR antagonists in both untreated and agonist‐treated MOR cells. CHO cells expressing the cloned human mu receptor (hMOR‐CHO cells) were incubated for 20 h with medium (control) or 10 μM (2 S ,4a R ,6a R ,7 R ,9 S ,10a S ,10b R )‐9‐(benzoyloxy)‐2‐(3‐furanyl)dodecahydro‐6a,10b‐dimethyl‐4,10‐dioxo‐2 H ‐naphtho‐[2,1‐ c ]pyran‐7‐carboxylic acid methyl ester (herkinorin, HERK). HERK treatment generates a high degree of basal signaling and enhances the ability to detect inverse agonists. [ 35 S]‐GTP‐γ‐S assays were conducted using established methods. We screened 21 MOR “antagonists” using membranes prepared from HERK‐treated hMOR‐CHO cells. All antagonists, including CTAP and 6β‐naltrexol, were inverse agonists. However, LTC‐274 ((−)‐3‐cyclopropylmethyl‐2,3,4,4α,5,6,7,7α‐octahydro‐1H‐benzofuro[3,2‐e]isoquinolin‐9‐ol)) showed the lowest efficacy as an inverse agonist, and, at concentrations less than 5 nM, had minimal effects on basal [ 35 S]‐GTP‐γ‐S binding. Other efforts in this study identified KC‐2‐009 ((+)‐3‐((1 R ,5 S )‐2‐(( Z )‐3‐phenylallyl)‐2‐azabicyclo[3.3.1]nonan‐5‐yl)phenol hydrochloride) as an inverse agonist at untreated MOR cells. In HERK‐treated cells, KC‐2‐009 had the highest efficacy as an inverse agonist. In summary, we identified a novel and selective MOR inverse agonist (KC‐2‐009) and a novel MOR antagonist (LTC‐274) that shows the least inverse agonist activity among 21 MOR antagonists. LTC‐274 is a promising lead compound for developing a true MOR neutral antagonist. Synapse 64:280–288, 2010. © 2009 Wiley‐Liss, Inc.