LA JOLLA, CA -- March 21, 2012 -- Scientists have for the first time determined the three-dimensional atomic structure of a human opioid receptor, a molecule on the surface of brain cells that binds to opioids and is centrally involved in pleasure, pain, addiction, depression, psychosis, and related conditions. Dozens of legal and illegal drugs, from heroin to hospital anesthetics, work by targeting these receptors. The detailed atomic structure information paves the way for the design of safer and more effective opioid drugs.
"This finding is going to have a major impact on understanding the fundamental principles of opioid receptor recognition and evolution," said Raymond Stevens, PhD, a professor at The Scripps Research Institute. Stevens is the senior author of the new study, which appears online in the journal Nature on March 21, 2012.
A Symphony of Activity
Opioid receptor subtypes in the human brain work together in a symphony of activity that is still not fully understood. The "mu" opioid receptors mediate feelings of pleasure and pain-relief; they are the prime targets of the body's own endorphin neurotransmitters as well as heroin, morphine, and most other opioid drugs. By contrast, "kappa" opioid receptors are bound by neurotransmitters known as dynorphins, and when activated can depress mood and produce dissociative, psychedelic experiences. The plant Salvia divinorum, which was originally cultivated by Mesoamerican societies for religious ceremonies and is now used widely as a recreational drug, has an active ingredient, Salvinorin A, that binds selectively and with high affinity to kappa opioid receptors.
"We don't know why kappa receptors evolved, but we know that they have been around for a long time in evolutionary terms; even frogs have them," said Bryan Roth, a professor of pharmacology and an opioid receptor expert at the University of North Carolina, whose group teamed with the Stevens lab for the new study.
If their psychedelic and mood-darkening effects could be avoided somehow, kappa opioid receptor activators, or "agonists," could be very useful medically. In animal studies, they act as mild and non-addicting pain-relievers, weaken the addictive effects of other drugs, and reduce irritable bowel signs. "Antagonist" compounds that block kappa opioid receptor activity also show promise as treatments for depression, anxiety, and other psychiatric conditions. Even the psychedelic effects associated with kappa receptor activation could be useful in providing insights into perception and consciousness. "This is a receptor that is important for how we see reality," said Roth.
In Pursuit of Missing Information
Not knowing the structural details of the kappa opioid receptor has made it hard for scientists to understand how the receptor works naturally and to design drugs that target it in the right ways. There are currently no FDA-approved drugs that bind selectively to kappa opioid receptors, either as agonists or antagonists.
To get these structural details, Scripps Research graduate student Huixian Wu, who was first author of the paper, first produced kappa opioid receptors in insect cells and used special techniques to keep the fragile receptor molecules stable in a single conformation. Working with Scripps Research Assistant Professor Vadim Cherezov, PhD, Wu was then able to crystallize and collect x-ray data that eventually led to the structure. An essential part of this stabilizing process involved the attachment of a suitable "ligand," a pharmacological molecule that binds the receptor tightly. "We tried a lot of different ligands, and Bryan suggested that we try JDTic, an experimental kappa opioid receptor antagonist," said Stevens. "It worked well in stabilizing the receptor, and once we discovered that, everything else quickly fell into place."
By X-raying such a crystallized protein complex from various angles, researchers can calculate a detailed three-dimensional model of its atomic structure. In this case, the X-ray data yielded a model with a resolution of about 2.9 angstroms, or 290 trillionths of a meter. The Stevens and Roth labs then designed and performed site-specific mutagenesis analyses in which they altered various residues of the receptor to better understand how its structure related to its function. Scripps Research Assistant Professor Seva Katrich, PhD, together with colleagues at Research Triangle Institute and Virginia Commonwealth University, then performed molecular modeling and docking analyses to further the understanding of the receptor-ligand interactions.
"This receptor's binding pocket is much bigger and deeper than any other we've studied; that may explain why so many different types of ligands bind to this receptor. A lot of work remains, though, in understanding opioid selectivity. As with the other receptor structures, this is just the beginning and we will see follow up studies take the understanding even further," said Stevens. He and his colleagues used their structural model to learn new details about the kappa receptor binding characteristics of Salvinorin A plus an array of experimental drugs.
With the publication of the receptor structure, scientists and drug companies also will be able to use the data to improve existing kappa-targeting compounds and design entirely new ones. "I can tell you, from e-mails and other conversations I've had recently, that people in the pharma industry are very eager to see this structural data," said Roth.
The Power of Collaboration
The kappa opioid receptor structure is the latest product of two interlinked, National Institutes of Health (NIH)-funded networks of laboratories, both based at Scripps Research.
The Joint Center for Innovative Membrane Protein Technologies, supported by the NIH Common Fund, develops improved techniques for the expression, purification, and structural characterization of membrane proteins and then disseminates those technologies rapidly to the broad scientific community. The GPCR Network, part of the Protein Structure Initiative (PSI:Biology) supported by the NIH's National Institute of General Medical Sciences (NIGMS), uses those techniques to determine the structures of human G-Protein Coupled Receptors (GPCRs) in close collaboration with the scientific community. GPCRS are the largest family of cell receptors and the one that includes opioid receptors. Of the nearly 800 human GPCRs for which scientists hope to develop atomic structural models, the kappa opioid receptor is only the eleventh published so far, but that number is rapidly changing. Last month, the Stevens laboratory published the structure of the S1P1 lipid receptor and the NMR characterization of the beta 2 adrenergic receptor, both in the journal Science.
"PSI:Biology promotes collaborative research between structural scientists and biologists to address important biological problems," said Jean Chin, PhD, who helps oversee PSI:Biology at the NIGMS. "The determination of the human kappa-opioid receptor structure bound to a compound with potential therapeutic value may lead to the design of more effective pain killers and anti-addiction medicines, demonstrating the value of these collaborations."
In addition to NIGMS and the NIH Common Fund, the study was supported by the National Institute of Mental Health Psychoactive Drug Screening Program in Chapel Hill, North Carolina, the National Institute of Drug Abuse (NIDA), and the NIDA Drug Supply Program for supplying opioid ligands.
In addition to Stevens, Roth, Wu, Cherezov, and Katrich, authors of the paper, "Structure of the human kappa opioid receptor in complex with JDTic," included Daniel Wacker, Mauro Mileni, Gye Won Han, Wei Liu, and Aaron A. Thompson of Scripps Research; Eyal Vardy and Xi-Ping Huang of Roth's laboratory at UNC; F. Ivy Carroll and S. Wayne Mascarella at the Research Triangle Institute in Research Triangle Park, NC; and Richard B. Westkaemper and Philip D. Mosier of Virginia Commonwealth University.