Structure-signal relationships at κ-opioid receptor (KOR) revealed by bicyclo[2.2.2]octene enamide derivatives

κ-Opioid receptor (KOR) agonists are promising candidates for analgesic development, yet their clinical application is limited by on-target adverse effects such as sedation and dysphoria. Attenuating β-arrestin2 recruitment while retaining G-protein signaling has therefore attracted interest as a strategy for improving KOR agonist pharmacology. However, how subtle ligand structural changes alter receptor interactions and signaling outcomes remains incompletely understood. Here, we synthesized a series of bicyclo[2.2.2]octene enamide derivatives and examined their structure-signal relationships by systematically varying linker length and amide N-substitution. Functional characterization showed that these derivatives retained robust G_i protein activation (E_max = 80.4-103% relative to dynorphin A) while exhibiting attenuated β-arrestin2 recruitment (E_max = 12.6-61.8%). Notably, within the NH series, β-arrestin2 efficacy did not scale uniformly with linker length, whereas N-methylation did not produce a consistent effect across matched analogues. Induced-fit docking showed similar static binding poses among the NH derivatives despite their distinct β-arrestin2 efficacies. Molecular dynamics simulations, in contrast, revealed differences in dynamic ligand-receptor interactions and ligand positioning within the TM2-TM3 region, particularly involving Q115^2.60, D138^3.32, Y139^3.33, and Y320^7.43. These findings suggest that KOR signaling outcomes are associated not simply with ligand occupancy of the TM2-TM3 region but also with the specific dynamic interactions and positioning established within this region. Our results provide a structural framework for understanding how subtle ligand modifications can produce distinct KOR signaling profiles.