Mechanistic Insights into G Protein-Biased κ‑Opioid Receptor Signaling Using Dual-Charged Naltrexamine Amides

Opioids remain a cornerstone of pain management, but currently used therapeutics are associated with serious side effects. While κ-opioid receptor (KOR) agonists offer an alternative to classical μ-opioid receptor (MOR) agonists, their clinical potential is limited by severe adverse effects. G protein-biased KOR agonists are a promising strategy for developing safer analgesics. In this study, we used virtual screening to develop novel dual-charged naltrexamine amide derivatives as tool compounds for investigating biased agonism at the KOR. All of the predicted ligands demonstrate low-nanomolar activity and G protein bias at both the KOR and MOR. Molecular dynamics simulations revealed a key allosteric communication involving TM4, TM5, and ICL2. These compounds achieve their effects through interactions with residues E209ECL2, D2235.35, E2976.58, and K2275.39. These findings provide insight into the structural mechanisms of KOR signaling bias and inform the rational design of improved KOR therapeutics.