Binding preference at the µ-opioid receptor underlies distinct pharmacology of cyclopropyl versus valeryl analogs of fentanyl.

Illicitly manufactured fentanyl is driving the current opioid crisis, and various fentanyl analogs are appearing in recreational drug markets worldwide. To assess the potential health risks posed by fentanyl analogs, it is necessary to understand structure-activity relationships for these compounds. Here we compared the pharmacology of two structurally related fentanyl analogs implicated in opioid overdose: cyclopropylfentanyl and valerylfentanyl. Cyclopropylfentanyl has a three-carbon ring attached to the carbonyl group on the fentanyl scaffold, whereas valerylfentanyl has a four-carbon chain at the same position. In vitro assays examining µ-opioid receptor (MOR) coupling to G proteins in CHO cells showed that cyclopropylfentanyl is a full agonist (EC50 = 8.6 nM, %Emax = 113%), with potency and efficacy similar to fentanyl (EC50 = 10.3 nM, %Emax = 113%). By contrast, valerylfentanyl is a partial agonist at MOR (EC50 = 179.8 nM, %Emax = 60%). Similar results were found in assays assessing MOR-mediated ß-arrestin recruitment in HEK cells. In vivo studies in male CD-1 mice demonstrated that both fentanyl analogs induce naloxone-reversible antinociception and respiratory suppression, but cyclopropylfentanyl is 100-times more potent as an antinociceptive agent (ED50 = 0.04 mg/kg, s. c.) than valerylfentanyl (ED50 = 4.0 mg/kg, s. c.). Molecular simulation results revealed that the alkyl chain of valerylfentanyl cannot be well accommodated by the active state of MOR and may transition the receptor toward an inactive state, converting the fentanyl scaffold to a partial agonist. Taken together, our results suggest that cyclopropylfentanyl presents much greater risk of adverse effects when compared to valerylfentanyl. Moreover, the summed findings may provide clues to the design of therapeutic opioids with reduced adverse side effects.

Alternatively spliced mu opioid receptor C termini impact the diverse actions of morphine.

Extensive 3' alternative splicing of the mu opioid receptor gene OPRM1 creates multiple C-terminal splice variants. However, their behavioral relevance remains unknown. The present study generated 3 mutant mouse models with truncated C termini in 2 different mouse strains, C57BL/6J (B6) and 129/SvEv (129). One mouse truncated all C termini downstream of Oprm1 exon 3 (mE3M mice), while the other two selectively truncated C-terminal tails encoded by either exon 4 (mE4M mice) or exon 7 (mE7M mice). Studies of these mice revealed divergent roles for the C termini in morphine-induced behaviors, highlighting the importance of C-terminal variants in complex morphine actions. In mE7M-B6 mice, the exon 7-associated truncation diminished morphine tolerance and reward without altering physical dependence, whereas the exon 4-associated truncation in mE4M-B6 mice facilitated morphine tolerance and reduced morphine dependence without affecting morphine reward. mE7M-B6 mutant mice lost morphine-induced receptor desensitization in the brain stem and hypothalamus, consistent with exon 7 involvement in morphine tolerance. In cell-based studies, exon 7-associated variants shifted the bias of several mu opioids toward ß-arrestin 2 over G protein activation compared with the exon 4-associated variant, suggesting an interaction of exon 7-associated C-terminal tails with ß-arrestin 2 in morphine-induced desensitization and tolerance. Together, the differential effects of C-terminal truncation illustrate the pharmacological importance of OPRM1 3' alternative splicing.