Table 1 In vitro pharmacological profile of NOP/MOP bifunctional agonists in binding and functional assays at the opioid receptors.

Details of the experimental procedures are given in Supplementary Materials and Methods. Values are means ± SEM of three independent experiments run in triplicate. NT, not tested (compounds with binding affinity Ki >100 nM were not tested in the functional assay); DPDPE, (d-penicillamine2,d-penicillamine5)-enkephalin.

Receptor binding Ki (nM)NOP functional assays*MOP functional assays*KOP functional assays*
[35S]GTPγS NOP[35S]GTPγS MOP[35S]GTPγS KOP
NOPMOPKOPDOPEC50 (nM)% StimEC50 (nM)% StimEC50 (nM)% Stim
N/OFQ0.08 ± 0.03133 ± 30247 ± 3.42846 ± 5124.0 ± 0.1100>10,000>10,000
DAMGO2.96 ± 0.5432.6 ± 4.06100
DPDPE1.11 ± 0.07
U69,5931.05 ± 0.0260.14 ± 7.45100
111.7 ± 3.75130.3 ± 0.7500.3 ± 28331.5 ± 1.6181.7 ± 4.428.2 ± 0.6>10,000NT
20.99 ± 0.0467.24 ± 2.5260.3 ± 221603 ± 63186 ± 4542.1 ± 1.0>10,000NT
30.84 ± 0.0630.8 ± 2.568.9 ± 3.2834 ± 74229 ± 2234.1 ± 0.4128 ± 139.3 ± 2.236.8 ± 11.721.35 ± 3.4
48.61 ± 0.5628.08 ± 2.769.7 ± 2243.09 ± 18.6123.5 ± 5027.5 ± 3.096 ± 38.78.2 ± 2.2>10,000
5 (AT-121)3.67 ± 1.1016.49 ± 2.1301.3 ± 35.4145.6 ± 25.534.7 ± 6.2941.1 ± 0.319.6 ± 6.914.2 ± 0.40NT
66.44 ± 0.4453.94 ± 2.0455.5 ± 63184.7 ± 63173.4 ± 2245.1 ± 12115.6 ± 206.7 ± 0>10,000
Morphine>10,0001.1 ± 0.146.9 ± 14.5140.0 ± 1.5>10,0005.4 ± 115.6 ± 0.593 ± 9576 ± 81.525.0 ± 1.95
Buprenorphine140 ± 11.20.15 ± 0.132.50 ± 1.26.13 ± 0.4>10,00012.6 ± 67.2 ± 3.527.0 ± 4.8>10,000

*Functional activity was determined by stimulation of [35S]GTPγS binding to cell membranes, and % stimulation was obtained as a percentage of stimulation of the standard full agonists, N/OFQ (for NOP), DAMGO (for MOP), and U69,593 (for KOP), which showed at least two- to five-fold stimulation over basal, indicative of a robust assay. The stimulation by the standard full agonists was taken as 100% when comparing stimulation by the test compound.