The goal of this proposal is to determine the structural basis for opioid receptor pharmacology and function. Opioid receptors constitute the major and the most effective target for the treatment of pain. The use of opioid drugs acting at these receptors is however a leading cause of death by overdose in Europe and North America. Both beneficial and adverse effects of illicit opioid drugs (opium, heroin) as well as approved therapeutics (morphine and codeine) are mediated by the activation of mu opioid receptor (?-OR), a member of the G protein-coupled receptors (GPCRs) superfamily. To understand the structural basis for ?-OR function, we obtained the first high-resolution crystal structure of an inactive state of this receptor along with a closely relate family member the delta-opioid receptor (?-OR). These structures provide new insights into OR preference for specific antagonist drugs but do not address important questions regarding OR activation mechanisms and, in particular, opioid drug efficacy. We therefore propose to characterize the structural basis of opioid receptor activation using a combination of biochemical and biophysical approaches.
Specific Aims i nclude:
Aim 1. Determine active state structures of ORs The inactive state structures of the ?-OR and ?-OR provided the first structural insights into the binding mode of morphinan antagonists. The goal of this aim is to obtain structural insights into the process of opioid receptor activation of the G protein Gi. We will initially focus on the use of crystallography and single particle electron microscopy to obtain three-dimensional structures of the ?-OR-Gi and ?-OR-Gi complexes. We will also develop a panel of camelid antibody fragments (nanobodies) that stabilized ligand-specific conformational states for crystallography. These will be used to determine the structural basis for the different functional properties of opioid receptor agonists.
Aim 2. Conformational dynamics of OR structure and activation. OR activation and more generally GPCR activation involves a complex allosteric coupling between ligand binding and G protein coupling domains that is poorly understood. Crystal structures offer a limited number of static snapshots of this dynamic process. We therefore propose to develop and apply biophysical approaches to characterize the structural plasticity and dynamic properties of ORs and to determine how this is translated into signaling complexity and ligand efficacy. Inactive state crystal structures will constitute an important starting point for designing and interpreting biochemical and biophysical studies, some of which include fluorescence, EPR and NMR spectroscopy. These studies will provide new insights into opioid ligand efficacy and the differences between small molecule and peptide ligands.

Public Health Relevance

The goal of this proposal is to determine the structural basis for opioid receptor pharmacology and function. Opioid receptors constitute the major and the most effective target for the treatment of acute and chronic pain. The use of opioid drugs acting at these receptors is, however, a leading cause of death by overdose in North America and Europe. This proposal will provide structural insights that will facilitate the development of safe and more effective drugs for opioid receptors.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DA036246-02
Application #
8677861
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Rapaka, Rao
Project Start
2013-06-15
Project End
2017-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
2
Fiscal Year
2014
Total Cost
$453,987
Indirect Cost
$83,791
Name
Stanford University
Department
Biophysics
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305