Heng Xu

Salvinorin A: Allosteric Interactions at the μ-Opioid Receptor

Salvinorin A [(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(acetyloxy)-2-(3-furanyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2h-naphtho[2,1-c]pyran-7-carboxylic acid methyl ester] is a hallucinogenic κ-opioid receptor agonist that lacks the usual basic nitrogen atom present in other known opioid ligands. Our first published studies indicated that Salvinorin A weakly inhibited μ-receptor binding, and subsequent experiments revealed that Salvinorin A partially inhibited μ-receptor binding. Therefore, we hypothesized that Salvinorin A allosterically modulates μ-receptor binding. To test this hypothesis, we used Chinese hamster ovary cells expressing the cloned human opioid receptor. Salvinorin A partially inhibited [3H]Tyr-d-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO) (0.5, 2.0, and 8.0 nM) binding with EMAX values of 78.6, 72.1, and 45.7%, respectively, and EC50 values of 955, 1124, and 4527 nM, respectively. Salvinorin A also partially inhibited [3H]diprenorphine (0.02, 0.1, and 0.5 nM) binding with EMAX values of 86.2, 64, and 33.6%, respectively, and EC50 values of 1231, 866, and 3078 nM, respectively. Saturation binding studies with [3H]DAMGO showed that Salvinorin A (10 and 30 μM) decreased the μ-receptor Bmax and increased the Kd in a dose-dependent nonlinear manner. Saturation binding studies with [3H]diprenorphine showed that Salvinorin A (10 and 40 μM) decreased the μ-receptor Bmax and increased the Kd in a dose-dependent nonlinear manner. Similar findings were observed in rat brain with [3H]DAMGO. Kinetic experiments demonstrated that Salvinorin A altered the dissociation kinetics of both [3H]DAMGO and [3H]diprenorphine binding to μ receptors. Furthermore, Salvinorin A acted as an uncompetitive inhibitor of DAMGO-stimulated guanosine 5′-O-(3-[35S]thio)-triphosphate binding. Viewed collectively, these data support the hypothesis that Salvinorin A allosterically modulates the μ-opioid receptor.

14-Alkoxy- and 14-acyloxypyridomorphinans: μ agonist/δ antagonist opioid analgesics with diminished tolerance and dependence side effects.

In the search for opioid ligands with mixed functional activity, a series of 5-(4-chlorophenyl)-4,5α-epoxypyridomorphinans possessing alkoxy or acyloxy groups at C-14 was synthesized and evaluated. In this series, the affinity and functional activity of the ligands were found to be influenced by the nature of the substituent at C-14 as well as by the substituent at N-17. Whereas the incorporation of a 3-phenylpropoxy group at C-14 on N-methylpyridomorhinan gave a dual MOR agonist/DOR agonist 17h, its incorporation on N-cyclopropylmethylpyridomorphinan gave a MOR agonist/DOR antagonist 17d. Interestingly, 17d, in contrast to 17h, did not produce tolerance or dependence effects upon prolonged treatment in cells expressing MOR and DOR. Moreover, 17d displayed greatly diminished analgesic tolerance as compared to morphine upon repeated administration, thus supporting the hypothesis that ligands with MOR agonist/DOR antagonist functional activity could emerge as novel analgesics devoid of tolerance, dependence, and related side effects.

Identification of a novel “almost neutral” μ‐opioid receptor antagonist in CHO cells expressing the cloned human μ‐opioid receptor

The basal (constitutive) activity of G protein‐coupled receptors allows for the measurement of inverse agonist activity. Some competitive antagonists turn into inverse agonists under conditions where receptors are constitutively active. In contrast, neutral antagonists have no inverse agonist activity, and they block both agonist and inverse agonist activity. The μ‐opioid receptor (MOR) demonstrates detectable constitutive activity only after a state of dependence is produced by chronic treatment with a MOR agonist. We therefore sought to identify novel MOR inverse agonists and novel neutral MOR antagonists in both untreated and agonist‐treated MOR cells. CHO cells expressing the cloned human mu receptor (hMOR‐CHO cells) were incubated for 20 h with medium (control) or 10 μM (2S,4aR,6aR,7R,9S,10aS,10bR)‐9‐(benzoyloxy)‐2‐(3‐furanyl)dodecahydro‐6a,10b‐dimethyl‐4,10‐dioxo‐2H‐naphtho‐[2,1‐c]pyran‐7‐carboxylic acid methyl ester (herkinorin, HERK). HERK treatment generates a high degree of basal signaling and enhances the ability to detect inverse agonists. [35S]‐GTP‐γ‐S assays were conducted using established methods. We screened 21 MOR “antagonists” using membranes prepared from HERK‐treated hMOR‐CHO cells. All antagonists, including CTAP and 6β‐naltrexol, were inverse agonists. However, LTC‐274 ((−)‐3‐cyclopropylmethyl‐2,3,4,4α,5,6,7,7α‐octahydro‐1H‐benzofuro[3,2‐e]isoquinolin‐9‐ol)) showed the lowest efficacy as an inverse agonist, and, at concentrations less than 5 nM, had minimal effects on basal [35S]‐GTP‐γ‐S binding. Other efforts in this study identified KC‐2‐009 ((+)‐3‐((1R,5S)‐2‐((Z)‐3‐phenylallyl)‐2‐azabicyclo[3.3.1]nonan‐5‐yl)phenol hydrochloride) as an inverse agonist at untreated MOR cells. In HERK‐treated cells, KC‐2‐009 had the highest efficacy as an inverse agonist. In summary, we identified a novel and selective MOR inverse agonist (KC‐2‐009) and a novel MOR antagonist (LTC‐274) that shows the least inverse agonist activity among 21 MOR antagonists. LTC‐274 is a promising lead compound for developing a true MOR neutral antagonist. Synapse 64:280–288, 2010.