Table 1.
Screened clinical opioids and receptor targets.
Table 2.
Optimized conditions for radioligand binding and functional assays.
Table 3.
Cell line saturation binding results.
Fig 1.
Summary concentration-response curves for radioligand binding.
Competition radioligand binding was performed as described in the Materials and Methods for all 9 clinical opioids and a positive control competitor ligand at all 9 receptor targets. Positive control: MOR/DOR/KOR = naloxone, NOP = nociceptin, CB1 = WIN55,212, σ1R = BD1008, NET/SERT/DAT = S-duloxetine. The curves shown here are reported as the mean ± SEM of the mean values calculated separately from N ≥ 3 independent experiments performed in duplicate. The data is further reported as the % of maximum binding, which is determined from only radioligand present and no competitor (100%) and as the non-specific binding, which is radioligand in the presence of 10 μM positive control compound (0%).
Table 4.
Competition radioligand binding affinity values.
Fig 2.
Summary concentration-response curves for receptor functional assays.
35S-GTPγS coupling was performed for the MOR, DOR, KOR, NOP, and CB1 in agonist mode and a transport uptake assay was performed for NET, SERT, and DAT, all as described in the Materials and Methods. All 9 clinical opioids and a positive control agonist or transport inhibitor was tested against 8 receptor targets (minus the σ1R). Positive control: MOR = endomorphin-2, DOR = DPDPE, KOR = U50,488, NOP = nociceptin, CB1 = WIN55,212, NET/DAT = S-duloxetine, SERT = GBR12909. The data was normalized to the stimulation caused by positive control compound (100%) and vehicle (0%). The data was further reported as the mean ± SEM of the mean values calculated independently from N ≥ 3 independent experiments performed in duplicate.
Table 5.
Functional assay potency and efficacy values.
Fig 3.
Selected opioid compounds tested as KOR antagonists.
Selected clinical opioids and a naloxone positive control were tested as antagonists vs. 100 nM U50,488 at the KOR using 35S-GTPγS coupling. A) The data was normalized to the stimulation caused by 100 nM U50,488 (100%) or vehicle (0%) and reported as the mean ± SEM of N = 3 independent experiments. B) The resulting potency (IC50) and efficacy (IMAX) values are reported as the mean ± SEM. The IMAX is normalized to the inhibition caused by the positive control naloxone (100%). Buprenorphine shows high efficacy and very high potency KOR antagonist activity, but the other clinical opioids show no evidence of antagonist activity.
Table 6.
Summary of novel drug and target interactions.
Fig 4.
The monoamine transporter inhibitor duloxetine causes a buprenorphine-selective enhancement of tail flick anti-nociception.
Male CD-1 mice were tested for tail flick anti-nociception baselines with 52°C water (10 second cutoff). The mice were then injected with duloxetine (20 mg/kg) or vehicle (1% Tween80 in saline) by the intraperitoneal route for 10 minutes, followed by subcutaneous buprenorphine (0.2 mg/kg), oxymorphone (0.3 mg/kg), or vehicle (saline). Tail flick latencies were then recorded over a 2 hour time course. Data reported as the latencies in raw seconds, mean ± SEM. Sample sizes of individual mice/group are noted in the graph legends. The Vehicle/Vehicle and Duloxetine/Vehicle groups were performed as one technical replicate. All other groups were performed as 2 technical replicates, with N = 5/group for each replicate. The same blinded experimenter performed all experiments. A) Vehicle or duloxetine alone had no effect on tail flick baselines. Duloxetine increased buprenorphine anti-nociception with an AUC increase of 50.6%. *, ** = p < 0.05, 0.01 vs. same time point Veh/Bupe group by 2 Way ANOVA with Fisher’s Least Significant Difference post-hoc test. B) Duloxetine had no effect on oxymorphone anti-nociception. p > 0.05.