Separation of binding affinity and intrinsic activity of the potent mu-opioid 14-methoxymetopon.

Receptor binding studies of 5,14-O-dimethyloxymorphone (14-methoxymetopon) in brain membranes have established its high affinity for mu-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 [(3)H]14-methoxymetopon for mu sites in calf striatal membranes and for a number of full-length splice variants of the cloned murine mu-opioid receptor 1 (mMOR-1) in transfected cell lines. The binding affinity of [(3)H]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-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assays with the MOR-1 variants, 14-methoxymetopon and the mu-opioid peptide [d-Ala(2),N-Me-Phe(4),Gly(5)-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 [(35)S]GTPgammaS 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.