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 | ||||||||
| NOP | MOP | KOP | DOP | EC50 (nM) | % Stim | EC50 (nM) | % Stim | EC50 (nM) | % Stim | |
| N/OFQ | 0.08 ± 0.03 | 133 ± 30 | 247 ± 3.4 | 2846 ± 512 | 4.0 ± 0.1 | 100 | >10,000 | >10,000 | ||
| DAMGO | 2.96 ± 0.54 | 32.6 ± 4.06 | 100 | |||||||
| DPDPE | 1.11 ± 0.07 | |||||||||
| U69,593 | 1.05 ± 0.02 | 60.14 ± 7.45 | 100 | |||||||
| 1 | 11.7 ± 3.75 | 130.3 ± 0.7 | 500.3 ± 28 | 331.5 ± 1.6 | 181.7 ± 4.4 | 28.2 ± 0.6 | >10,000 | — | NT | |
| 2 | 0.99 ± 0.04 | 67.24 ± 2.5 | 260.3 ± 22 | 1603 ± 63 | 186 ± 45 | 42.1 ± 1.0 | >10,000 | — | NT | |
| 3 | 0.84 ± 0.06 | 30.8 ± 2.5 | 68.9 ± 3.2 | 834 ± 74 | 229 ± 22 | 34.1 ± 0.4 | 128 ± 13 | 9.3 ± 2.2 | 36.8 ± 11.7 | 21.35 ± 3.4 |
| 4 | 8.61 ± 0.56 | 28.08 ± 2.7 | 69.7 ± 22 | 43.09 ± 18.6 | 123.5 ± 50 | 27.5 ± 3.0 | 96 ± 38.7 | 8.2 ± 2.2 | >10,000 | — |
| 5 (AT-121) | 3.67 ± 1.10 | 16.49 ± 2.1 | 301.3 ± 35.4 | 145.6 ± 25.5 | 34.7 ± 6.29 | 41.1 ± 0.3 | 19.6 ± 6.9 | 14.2 ± 0.40 | NT | |
| 6 | 6.44 ± 0.44 | 53.94 ± 2.0 | 455.5 ± 63 | 184.7 ± 63 | 173.4 ± 22 | 45.1 ± 12 | 115.6 ± 20 | 6.7 ± 0 | >10,000 | — |
| Morphine | >10,000 | 1.1 ± 0.1 | 46.9 ± 14.5 | 140.0 ± 1.5 | >10,000 | 5.4 ± 1 | 15.6 ± 0.5 | 93 ± 9 | 576 ± 81.5 | 25.0 ± 1.95 |
| Buprenorphine | 140 ± 11.2 | 0.15 ± 0.13 | 2.50 ± 1.2 | 6.13 ± 0.4 | >10,000 | 12.6 ± 6 | 7.2 ± 3.5 | 27.0 ± 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.