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NEUROPHARMACOLOGY

Separation of Binding Affinity and Intrinsic Activity of the Potent µ-Opioid 14-Methoxymetopon

Laboratory of Molecular Neuropharmacology, Memorial Sloan-Kettering Cancer Center, New York, New York (L.M., C.G., J.M., G.W.P.); Department of Pharmaceutical Chemistry, Institute of Pharmacy and Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, Austria (H.S.); and Institute of Biochemistry, Biological Research Center of Hungarian Academy of Sciences, Szeged, Hungary (G.T.)

Receptor binding studies of 5,14-O-dimethyloxymorphone (14-methoxymetopon) in brain membranes have established its high affinity for µ-binding sites, but its analgesic potency far exceeds the modest increase in binding affinity relative to other opioids. The current study has established the selectivity of [³H]14-methoxymetopon for µ sites in calf striatal membranes and for a number of full-length splice variants of the cloned murine µ-opioid receptor 1 (mMOR-1) in transfected cell lines. The binding affinity of [³H]14-methoxymetopon for the variants expressed in Chinese hamster ovary cells was quite high, with K_D values around 0.2 nM for all of the variants with the exception of mMOR-1F (K_D of 1.2 nM). The affinity for most of the expressed variants was greater than that seen in the brain membranes (K_D of 0.99 nM). Functionally, in guanosine 5'-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPS) binding assays with the MOR-1 variants, 14-methoxymetopon and the µ-opioid peptide [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO) showed similar efficacies, as determined by maximal stimulation, but 14-methoxymetopon was up to 65-fold more potent than DAMGO. The greatest difference was seen with mMOR-1E and the least with mMOR-1C, which displayed only a 10-fold difference. These potency differences in the stimulation of [³⁵S]GTPS binding far exceeded the differences in binding affinity. The differences between 14-methoxymetopon and DAMGO remained after normalizing the potency shifts based upon receptor binding affinities and varied from 1.2-fold with mMOR-1C to 21-fold for mMOR-1 and 42-fold with mMOR-1F. Thus, 14-methoxymetopon is a potent agonist against all of the mMOR-1 splice variants, but its potency ranged widely despite similar binding affinities for most of the variants and may give insight into its unusual pharmacological profile.

Address correspondence to: Dr. Gavril W. Pasternak, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021. E-mail: pasterng{at}mskcc.org