Abstract

The opioid receptor family is comprised of four G protein-coupled receptors, mu (MOR), kappa (KOR), delta (DOR), and nociceptin (NOP), which are involved in many physiological processes including pain, emotion, and digestion, making them important targets for drug development. MOR has commonly been used as an effective target for pain therapies. However, MOR-based therapeutics are associated with negative side effects, including addiction and respiratory depression, limiting their use in chronic pain treatments. Other receptors in the opioid family, particularly DOR, pose as potential alternative for chronic pain treatment. However, despite extensive research, the molecular mechanisms behind ligand binding, receptor activation, and transducer signaling remain poorly understood. To better understand DOR, we used cryo-electron microscopy (cryo-EM) and pharmacological assays to investigate processes underlying ligand pharmacology, aiding in the development of the next generation of DOR-based therapeutics. We used cryo-EM to solve structures of various ligands, including peptides, small molecule agonists, and antagonists, bound to DOR. These structures, together with pharmacological assays, provided insights into key residues responsible for DOR selectivity, a hydrophobic pocket relevant for receptor activation, and distinct residue conformations in peptide vs small molecule binding. Using insights from structure, we then developed the first selective DOR partial agonist using a bitopic approach, engaging two distinct pockets in the receptor. The reduced receptor activity of partial agonists allowed us to develop a ligand with analgesia and minimized side effects. Because our pharmacological experiments rely on ensemble measurements, which limit spatiotemporal resolution, we conducted single-molecule fluorescence resonance energy transfer (smFRET) experiments to study discrete measurements and gain deeper insights into receptor dynamics. smFRET experiments showed that, even in the presence of DOR full agonists and G proteins, the fully active-state DOR only occupied 60% of states, which is in contrast to the nearly 100% population observed at MOR. We have also extended the smFRET studies to KOR and observed a higher population of fully active state than DOR but a lesser extent than MOR. These data suggest that opioid receptors have intrinsically differing coupling efficiency with G proteins which could contribute to their effectiveness in pain modulation. Together, the results from this study provide a deeper understanding of opioid receptor function and lay out a framework for developing partial agonists with safer therapeutic profiles.

Committee Chair

Tao Che

Committee Members

Susruta Majumdar; Baron Chanda; Jose Moron-Concepcion; Sarah England

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Neurosciences)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

5-8-2025

Language

English (en)

Author's ORCID

https://orcid.org/0000-0001-8549-0413

Download

Available for download on Thursday, May 06, 2027

Included in

Biology Commons

Share

COinS