| Assay Method Information | |
| | Radioligand Binding Assay |
| Description: | For a primary screen of selected molecules, binding to OR was assessed by measuring competition against the radioligand 3H-diprenorphine (3H-DPN). Each compound was initially tested at 20 M and was incubated with 3H-DPN at a concentration equal to the Kd (0.4 nM) of the radioligand in OR containing Sf9 insect cell membranes. The reaction contained 40 fmol of OR and was incubated in a buffer of 20 mM HEPES pH 7.5, 100 mM sodium chloride, and 0.1% bovine serum albumin for 1 hour at 25 ° C. To separate free from bound radioligand, reactions were rapidly filtered over Whatman GF/B filters with the aid of a Brandel harvester and 3H-DPN counts were measured by liquid scintillation. Compounds with more than 25% of 3H-DPN radioactivity were further tested in full dose-response to determine the affinity (Ki) in HEK293 membranes. Subsequently, the 15 analogs were tested in full dose-response for affinity at the OR and the OR by the National Institutes of Mental Health Psychoactive Drug Screen Program (PDSP), as were the affinities of compounds 12, PZM21, and their stereoisomers at the OR, OR, OR and nociception receptor. |
| Affinity data for this assay | |
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