Posts tagged opioid receptors
Articles and news from the latest research reports.
Researchers Identify Brain Areas Activated by Itch-Relieving Drug
Areas of the brain that respond to reward and pleasure are linked to the ability of a drug known as butorphanol to relieve itch, according to new research led by Gil Yosipovitch, MD, Professor and Chair of the Department of Dermatology at Temple University School of Medicine (TUSM), and Director of the Temple Itch Center. The findings point to the involvement of the brain’s opioid receptors—widely known for their roles in pain, reward, and addiction—in itch relief, potentially opening up new avenues to the development of treatments for chronic itch.
The article, published online September 11, in the Journal of Investigative Dermatology, is the first to show precisely where in the brain butorphanol works to relieve itch. In identifying those areas, the study helps to explain why butorphanol works better for chronic itching mediated by histamine, a small molecule involved in allergic reactions, than for nonhistamine-related types of itch.
"The research allows us to assess butorphanol’s effects," Dr. Yosipovitch said. "We can now identify better targets in the brain that drugs can work on to relieve itch."
The research marks an important step toward the development of itch-specific agents. As Dr. Yosipovitch explained, chronic itching, which affects roughly 12 percent of the population, comprises not just one disease, but many—ranging from atopic eczema and psoriasis to systemic diseases such as lymphoma and chronic liver failure. Biochemically, each of those diseases induces itching via one of two main pathways: one that is mediated by histamine and one that is not. Most pathological itching originates along nonhistaminergic pathways.
Working with Alexandru D. P. Papoiu, MD, PhD, at Wake Forest University School of Medicine, Dr. Yosipovitch experimentally induced itch in human volunteers using either histamine or cowhage, which incites nonhistaminergic itching. Study volunteers were then treated with either butorphanol or a placebo and subjected to functional magnetic resonance imaging (fMRI) to analyze brain activity and assess the effects of butorphanol (or placebo). When volunteers returned seven days later, they received the other treatment and again underwent fMRI.
Butorphanol suppressed histamine itching in all cases and reduced cowhage itching in 35 percent of subjects. The drug’s suppression of histamine itching was associated specifically with the activation of brain areas known as the nucleus accumbens and septal nuclei—areas located deep at the base of the forebrain. The regions are notably rich in so-called kappa (κ)-opioid receptors, on which butorphanol acts. By contrast, the relief of cowhage itch by butorphanol was linked to effects in other brain areas.
The findings suggest that butorphanol works primarily on κ-opioid receptors to suppress the itch sensation induced by histamine. But the drug also has important effects on an itch pathway that does not involve histamine, where the demand for new treatments is greatest.
How nonhistaminergic itching is reduced through the involvement of opioid receptors remains unclear. Opioid receptors modulate the transmission of information about itch in the brain and occur in high levels in the areas of the brain that house neural pathways associated with reward. Reward pathways are known particularly for their response to pleasurable stimuli. Dr. Yosipovitch and Dr. Papoiu have shown in previous work that the activation of reward circuits is correlated with pleasurability and the degree of itch relief derived from self-scratching.
The new study, which Yosipovitch carried out at Wake Forest University prior to joining the TUSM faculty in 2013, further illustrates the power of applying imaging technologies to basic questions in itch research. At Temple’s Itch Center, Yosipovitch is continuing to explore those applications.
"We are in a position now to better understand the itch-scratch cycle," he said. "To break the cycle from the top down, knowing where to target receptors in the brain, would be a major achievement."
(Source: temple.edu)
A New Target for Alcoholism Treatment: Kappa Opioid Receptors
The list of brain receptor targets for opiates reads like a fraternity: Mu Delta Kappa. The mu opioid receptor is the primary target for morphine and endogenous opioids like endorphin, whereas the delta opioid receptor shows the highest affinity for endogenous enkephalins. The kappa opioid receptor (KOR) is very interesting, but the least understood of the opiate receptor family.
Until now, the mu opioid receptor received the most attention in alcoholism research. Naltrexone, a drug approved by the U.S. Food and Drug Administration for the treatment of alcoholism, acts by blocking opiate action at brain receptors and is most potent at the mu opioid receptor. In addition, research has suggested that a variant of the gene that codes for the mu opioid receptor (OPRM1) may be associated with the risk for alcoholism and the response to naltrexone treatment.
However, naltrexone also acts at the kappa opioid receptor and it has not been clear whether this effect of naltrexone is relevant to alcoholism treatment.
A growing body of research in animals implicates the KOR in alcoholism. Stimulation of the KOR, which occurs with alcohol intake, is thought to produce unpleasant and aversive effects. This receptor is hypothesized to play a role in alcohol dependence, at least in part, by promoting negative reinforcement processes. In other words, the theory postulates that during development of alcohol dependence, the KOR system becomes overstimulated, producing dysphoria and anhedonia, which then leads to further alcohol seeking and escalation of alcohol intake that serves to self-medicate those negative symptoms.
A new study in Biological Psychiatry, led by Dr. Brendan Walker at Washington State University, used a rat model of alcohol dependence to directly investigate the KOR system following chronic alcohol exposure and withdrawal.
They found that the KOR system is dysregulated in the amygdala of alcohol-dependent rats, a vital brain region with many functions, including regulation of emotional behavior and decision-making. Chronic alcohol consumption is known to cause neuroadaptations in the amygdala. In this study specifically, they found increased dynorphin A and increased KOR signaling in the amygdala of alcohol-dependent rats.
When the rats were in acute alcohol withdrawal, the researchers administered different drugs, each of which target the KOR system in precise ways, directly into the amygdala. Using this site-specific antagonism, they observed that alcohol dependence-related KOR dysregulation directly contributes to the excessive alcohol consumption that occurs during withdrawal.
“These data provide important new support for the hypothesis that kappa opioid receptor blockers might play a role in the treatment of alcoholism,” said Dr. John Krystal, Editor of Biological Psychiatry. “This study suggests that one role might be to prevent a relapse to alcohol use among patients recently withdrawn from alcohol.”
“This dataset demonstrates the extensive nature of the neuroadaptations the brain undergoes when chronically exposed to alcohol. The implications of these results are far reaching and should help guide pharmacotherapeutic development efforts for the treatment of alcohol use disorders,” said Walker. “Pharmacological compounds that alleviate the negative emotional / mood states that accompany alcohol withdrawal, by attenuating the excessive signaling in the dynorphin / kappa-opioid receptor system, should result in enhanced treatment compliance and facilitate the transition away from alcohol dependence.”
Additional extensive research will be necessary to identify and test the effectiveness of specific drugs that act on the KOR system, but these findings provide researchers with a potentially successful path forward to developing new drugs for the treatment of alcoholism.
Study identifies new drug target for chronic, touch-evoked pain
Researchers at the School of Medicine have identified a subset of nerve cells that mediates a form of chronic, touch-evoked pain called tactile allodynia, a condition that is resistant to conventional pain medication.
The discovery could point researchers to more fruitful efforts to develop effective drugs for the condition.
Touch-evoked pain occurs as part of a larger neuropathic pain condition arising from damage or disruption of nerve-cell circuits or signals caused by disorders such as alcoholism, diabetes, shingles and AIDS, or procedures such as spine surgery and chemotherapy. For patients with tactile allodynia, the slightest touch — a gentle caress or the brush of shirt against skin — can cause excruciating pain because changes in nerve-cell signals or networks trick the brain into mistaking touch for pain.
The study, published online Feb. 27 in Neuron, found that these “touch” neurons are different from the usual “pain” neurons that respond to stimuli such as cuts or bruises.
Unlike pain caused by such wounds, neuropathic pain is difficult to manage because little can be done to repair nerve damage. Managing it may require strong painkillers or combinations of treatments.
Common painkillers such as morphine have little effect on touch-evoked pain, possibly because they don’t target the touch neurons, the authors say. Morphine binds to specific protein-binding sites on pain neurons called mu opioid receptors, or MORs, and cuts off the their signals so that the brain can no longer sense pain.
However, the touch neurons do not carry MORs, which is why morphine cannot bind to them and block the pain. Instead, they carry delta opioid receptors, or DORs, whose role in pain control has been unclear until recently.
"That’s been the problem so far; any type of severe pain you have, you go into the clinic and very likely you will be treated with morphine-like opioids," said Gregory Scherrer, PharmD, PhD, the senior author of the study and an assistant professor of anesthesia. "You can give some of these patients as much morphine as you want; it won’t work if the mu opioid receptor is not present on the neurons that underlie that type of pain."
There are currently no Food and Drug Administration-approved pain-control drugs that target DORs. Previous attempts at developing DOR-targeting drugs haven’t succeeded because researchers didn’t know what type of pain such drugs would be useful for, Scherrer said.
Two DOR-binding drugs developed for knee pain by Adolor Corp., a biotechnology firm, for instance, probably failed because there is no compelling evidence that DOR was present or involved. AstraZeneca, another pharmaceutical firm, also had a DOR program but recently stopped its research efforts, Scherrer added.
"Now that we have provided a rationale and mechanism supporting the utility of DOR agonists for cutaneous pain and tactile allodynia, these companies will be able to design trials more carefully to evaluate specifically the drugs’ efficacy against touch-evoked pain," he said.
Earlier studies by Scherrer and others hinted at the presence of special nerve fibers on the skin that might contribute to touch-evoked pain.
In the current study, Scherrer and colleagues used fluorescent mouse models to isolate these neurons and identify how they control touch-evoked pain. They found that DOR can play an inhibitory role in these neurons: When proteins bind to DOR, they cut off communication to the spinal cord, through which sensory signals travel to the brain.
DOR-carrying “touch” neurons pervade the skin and could easily be targeted by drugs in the form of skin patches or topical creams, Scherrer suggested.
"By contrast, most MOR-carrying neurons penetrate internal organs," he said. "That’s why morphine is effective in treating post-surgery pain, for example."
Scherrer and fellow researchers tested two different DOR-binding compounds individually on mice and found that both reduced the mice’s sensitivity to touch-evoked pain.
Preliminary studies also indicate that DOR-targeting drugs might not cause dramatic side effects like morphine does, especially if they can be used topically, Scherrer said.
"Morphine and other MOR-targeting drugs have myriad deleterious side effects — including addiction, respiratory depression, constipation, nausea and vomiting — that further limits their utility for chronic pain management," he said.
The next step is to determine whether DOR could be a target for other types of pain, such as arthritis pain, pain from bone cancer and muscle pain, Scherrer added.
The findings also suggest that the body’s opioid system — normally associated with pain and addiction — may also respond to other stimuli such as touch.
"We may have underestimated the importance of the opioid system and what can be achieved with drugs targeting other subtypes of opioid receptors," Scherrer said.
(Source: med.stanford.edu)
Scientists Develop Promising Drug Candidates for Pain, Addiction
Scientists from the Florida campus of The Scripps Research Institute (TSRI) have described a pair of drug candidates that advance the search for new treatments for pain, addiction and other disorders.
The two new drug scaffolds, described in a recent edition of The Journal of Biological Chemistry, offer researchers novel tools that act on a demonstrated therapeutic target, the kappa opioid receptor (KOR), which is located on nerve cells and plays a role in the release of the neurotransmitter dopamine. While compounds that activate KOR are associated with positive therapeutic effects, they often also recruit a molecule known as βarrestin2 (beta arrestin), which is associated with depressed mood and severely limits any therapeutic potential.
“Compounds that act at kappa receptors may provide a means for treating addiction and for treating pain; however, there is the potential for the development of depression or dysphoria associated with this receptor target,” said Laura Bohn, a TSRI associate professor who led the study. “There is evidence that the negative feelings caused by kappa receptor drugs may be, in part, due to receptor actions through proteins called beta arrestins. Developing compounds that activate the receptors without recruiting beta arrestin function may serve as a means to improve the therapeutic potential and limit side effects.”
The new compounds are called “biased agonists,” activating the receptor without engaging the beta arrestins.
Research Associate Lei Zhou, first author of the study with Research Associate Kimberly M. Lovell, added, “The importance of these biased agonists is that we can manipulate the activation of one particular signaling cascade that produces analgesia, but not the other one that could lead to dysphoria or depression.”
The researchers note that the avoidance of depression is particularly important in addiction treatment, where depressed mood can play a role in relapse.
The two drug candidates also have a high affinity and selectivity for KOR over other opioid receptors and are able to pass through the blood-brain barrier. Given these promising attributes, the scientists plan to continue developing the compounds.
(Source: scripps.edu)
Scientists Solve 40-year Mystery of How Sodium Controls Opioid Brain Signaling
Scientists have discovered how the element sodium influences the signaling of a major class of brain cell receptors, known as opioid receptors. The discovery, from The Scripps Research Institute (TSRI) and the University of North Carolina (UNC), suggests new therapeutic approaches to a host of brain-related medical conditions.
“It opens the door to understanding opioid related drugs for treating pain and mood disorders, among others,” said lead author Dr. Gustavo Fenalti, a postdoctoral fellow in the laboratory of Professor Raymond C. Stevens of TSRI’s Department of Integrative Structural and Computational Biology.
“This discovery has helped us decipher a 40-year-old mystery about sodium’s control of opioid receptors,” said Stevens, who was senior author of the paper with UNC pharmacologist Professor Bryan Roth. “It is amazing how sodium sits right in the middle of the receptor as a co-factor or allosteric modulator.”
The findings appear in an advanced online publication in the journal Nature on January 12, 2014.
A Sharper Image
The researchers revealed the basis for sodium’s effect on signaling with a high-resolution 3-D view of an opioid receptor’s atomic structure. Opioid receptors are activated by peptide neurotransmitters (endorphins, dynorphins and enkephalins) in the brain. They can also be activated by plant-derived and synthetic drugs that mimic these peptides: among them morphine, codeine, oxycodone and heroin.
Despite these receptors’ crucial importance in health and disease, including pain disorders and addictions, scientists have only begun to understand in detail how they work. Opioid receptors are inherently flimsy and fragile when produced in isolation, and thus have been hard to study using X-ray crystallography, the usual structure-mapping method for large proteins.
In recent years, the Stevens laboratory has helped pioneer the structure determination of G protein-coupled receptors. Although the first crystallographic structures of opioid receptors were determined in 2012, these structural models weren’t fine-grained enough to solve a lingering mystery, particularly for the human delta opioid receptor.
That mystery concerned the role of sodium. The element is perhaps best known to biologists as one of the key “electrolytes” needed for the basic workings of cells. In the early 1970s, researchers in the laboratory of neuroscientist Solomon Snyder at Johns Hopkins University, who had helped discover opioid receptors, found evidence that sodium ions also act as a kind of switch on opioid receptor signaling. They noted that at concentrations normally found in brain fluid, these ions reduced the ability of opioid peptides and drugs like morphine to interact with opioid receptors.
How sodium could exert this indirect (“allosteric”) effect on opioid receptor activity was unclear—and has remained an unsolved puzzle for decades. Now that scientists have discovered the mechanism of sodium’s effect, then in principle they can exploit it to develop better opioid-receptor-targeting drugs.
A Switch Controlling Pain, Depression and Mood Disorders
For the new study, the team constructed a novel, fusion-protein-stabilized version of one of the main opioid receptors in the human brain, known as the delta opioid receptor, and managed to form crystals of it for X-ray crystallography. The latter revealed the receptor’s 3-D atomic structure to a resolution of 1.8 Angstroms (180 trillionths of a meter)—the sharpest picture yet of an opioid receptor.
“Such a high resolution is really necessary to be able to understand in detail how the receptor works,” said Stevens.
The analysis yielded several key details of opioid receptor structure and function, most importantly the details of the “allosteric sodium site,” where a sodium ion can slip in and modulate receptor activity.
The team was able to identify the crucial amino acids that hold the sodium ion in place and transmit its signal-modulating effect. “We found that the presence of the sodium ion holds the receptor protein in a shape that gives it a different affinity for its corresponding neurotransmitter peptides,” Fenalti said.
With the structural data in hand, the researchers designed new versions of the receptor, in which key sodium-site amino-acids were mutated, to see how this would affect receptor signaling. Co-lead author Research Associate Patrick M. Giguere and colleagues in Roth’s Laboratory at UNC, which has long collaborated with the Stevens laboratory, tested these mutant receptors and found that certain amino-acid changes cause radical shifts in the receptor’s normal signaling response.
The most interesting shifts involved a little-understood secondary or “alternative” signaling route, known as the beta-arrestin pathway, whose activity can have different effects depending on the type of brain cell involved. Some drugs that normally bind to the delta opioid receptor and have little or no effect on the beta-arrestin pathway turned out to strongly activate this pathway in a few of these mutant receptors.
In practical terms, these findings suggests a number of ways in which new drugs could target these receptors—and not only delta opioid receptors but also the other two “classical” opioid receptors, mu and kappa opioid receptors. “The sodium site architecture and the way it works seems essentially the same for all three of these opioid receptor types,” said Fenalti.
Nociceptin: Nature’s Balm for the Stressed Brain
Collaborating scientists at The Scripps Research Institute (TSRI), the National Institutes of Health (NIH) and the University of Camerino in Italy have published new findings on a system in the brain that naturally moderates the effects of stress. The findings confirm the importance of this stress-damping system, known as the nociceptin system, as a potential target for therapies against anxiety disorders and other stress-related conditions.
“We were able to demonstrate the ability of this nociceptin anti-stress system to prevent and even reverse some of the cellular effects of acute stress in an animal model,” said biologist Marisa Roberto, associate professor in TSRI’s addiction research department, known as the Committee on the Neurobiology of Addictive Disorders.
Roberto was a principal investigator for the study, which appears in the January 8, 2014 issue of the Journal of Neuroscience.
A Variety of Effects
Nociceptin, which is produced in the brain, belongs to the family of opioid neurotransmitters. But the resemblance essentially ends there. Nociceptin binds to its own specific receptors called NOP receptors and doesn’t bind well to other opioid receptors. The scientists who discovered it in the mid-1990s also noted that when nociceptin is injected into the brains of mice, it doesn’t kill pain—it makes pain worse.
The molecule was eventually named for this “nociceptive” (pain-producing) effect. However, subsequent studies demonstrated that, by activating its corresponding receptor NOP, nociceptin acted as an antiopioid and not only affected pain perception, but also blocked the rewarding properties of opioids such as morphine and heroin.
Perhaps of greatest interest, several studies in rodents have found evidence that nociceptin can act in the amygdala, a part of the brain that controls basic emotional responses, to counter the usual anxiety-producing effects of acute stress. There have been hints, too, that this activity occurs automatically as part of a natural stress-damping feedback response.
Scientists have wanted to know more about the anti-stress activity of the nociceptin/NOP system, in part because it might offer a better way to treat stress-related conditions. The latter are common in modern societies, including post-traumatic stress disorder as well as the drug-withdrawal stress that often defeats addicts’ efforts to kick the habit.
Reducing the Stress Reaction
For the new study, Roberto and her collaborators looked in more detail at the nociceptin/NOP system in the central amygdala.
First, Markus Heilig’s laboratory at the National Institute on Alcohol Abuse and Alcoholism (NIAAA), part of the NIH, measured the expression of NOP-coding genes in the central amygdala in rats. Heilig’s team found strong evidence that stress changes the activity of nociceptin/NOP in this region, indicating that the system does indeed work as a feedback mechanism to damp the effects of stress. In animals subjected to a standard laboratory stress condition, NOP gene activity rose sharply, as if to compensate for the elevated stress.
Roberto and her laboratory at TSRI then used a separate technique to measure the electrical activity of stress-sensitive neurons in the central amygdala. As expected, this activity rose when levels of the stress hormone CRF rose and started out at even higher levels in the stressed rats. But this stress-sensitive neuronal activity could be dialed down by adding nociceptin. The stress-blocking effect was especially pronounced in the restraint-stressed rats—probably due to their stress-induced increase in NOP receptors.
Finally, biologist Roberto Ciccocioppo and his laboratory at the University of Camerino conducted a set of behavioral experiments showing that injections of nociceptin specifically into the rat central amygdala powerfully reduced anxiety-like behaviors in the stressed rats, but showed no behavioral effect in non-stressed rats.
The three sets of experiments together demonstrate, said Roberto, that “stress exposure leads to an over-activation of the nociceptin/NOP system in the central amygdala, which appears to be an adaptive feedback response designed to bring the brain back towards normalcy.”
In future studies, she and her colleagues hope to determine whether this nociceptin/NOP feedback system somehow becomes dysfunctional in chronic stress conditions. “I suspect that chronic stress induces changes in amygdala neurons that can contribute to the development of some anxiety disorders,” said Roberto.
Compounds that mimic nociceptin by activating NOP receptors—but, unlike nociceptin, could be taken in pill form—are under development by pharmaceutical companies. Some of these appear to be safe and well tolerated in lab animals and may soon be ready for initial tests in human patients, Ciccocioppo said.
Sticks and stones: Brain releases natural painkillers during social rejection
Finding that the opioid system can act to ease social pain, not just physical pain, may aid understanding of depression and social anxiety
A brain image showing in orange/red one area of the brain where the natural painkiller (opioid) system was highly active in research volunteers who are experiencing social rejection. This region, called the amygdala, was one of several where the U-M team recorded the first images of this system responding to social pain, not just physical pain. Studying this response, and the variation between people, could aid understanding of depression and anxiety. Credited to UofM Health.
“Sticks and stones may break my bones, but words will never hurt me,” goes the playground rhyme that’s supposed to help children endure taunts from classmates. But a new study suggests that there’s more going on inside our brains when someone snubs us – and that the brain may have its own way of easing social pain.
The findings, recently published in Molecular Psychiatry by a University of Michigan Medical School team, show that the brain’s natural painkiller system responds to social rejection – not just physical injury.
What’s more, people who score high on a personality trait called resilience – the ability to adjust to environmental change – had the highest amount of natural painkiller activation.
(Source: uofmhealth.org)
Seeking new methods to treat heroin addiction
“Heroin itself is an inactive substance,” explains Jørg Mørland, Norwegian forensic medicine and toxicology researcher. “The substances that heroin forms in the body are mainly what enter the brain and cause the narcotic effects.”
The heroin high and feelings of pain relief manifest themselves almost immediately after the drug has been injected. Yet it was shown many years ago that heroin is inactive at the opioid receptors in the brain.
So what is it about heroin that brings about such a pronounced effect? A number of research projects funded under the Programme on Alcohol and Drug Research (RUSMIDDEL) at the Research Council of Norway may help to solve the mystery.
“Gaining a thorough understanding of the effects of heroin and of the neurobiological mechanisms involved will be a valuable basis for the development of new treatments for addiction,” states Jørg Mørland, who is the project manager of an ongoing project on this important subject, the most recent in a long line of such Norwegian projects which he has headed.
Dr Mørland is a senior researcher at the Norwegian Institute of Public Health and Professor emeritus at the University of Oslo. Through studies on rats and mice, he and his colleagues have come up with new findings that may be significant to the development of new treatment methods.
Heroin metabolises rapidly
One widely-held theory has been that heroin passes quickly into the brain where it is converted into morphine, and that what users are actually experiencing are the effects of morphine. As it turns out, however, heroin undergoes a number of important transformations on its way to the brain. Just a few minutes after injection, the conversion of heroin into the metabolite 6-MAM is almost complete.
“Our research shows that it is primarily 6-MAM that crosses the blood-brain barrier and that heroin as such only enters the brain to a small degree. Thirty minutes after injecting heroin, 6-MAM is the predominant substance both in the blood and in the brain,” Dr Mørland explains.
The presence of 6-MAM also results in a sharp increase in the signalling molecule, dopamine, in certain areas of the brain. This plays a pivotal role in the rewarding effect.
“This points towards 6-MAM as the main substance behind all the acute effects of heroin,” says Dr Mørland.
“After about an hour, most of the 6-MAM has been converted into morphine. Morphine acts rapidly on the body and is the dominant component for the next hours, but from six to twelve hours after injection the effects observed are mostly consequences of a metabolite formed from morphine, morphin-6-glucuronide.
Looking for a new treatment
“We are working to understand the roles of all these metabolites and to investigate potential treatments to counter their effects,” Dr Mørland states.
The current approach to treating heroin addiction in Norway is pharmacotherapy – using methadone, subutex or subuxone. These are synthetic substances that all work in the same way as heroin, however, and are addictive in their own right.
“The treatment method involves administering these substances over the course of a day to reduce the rewarding effect. The intent is to diminish the patient’s preoccupation with finding heroin in order to lead a more normal life,” Dr Mørland points out.
Researchers at the Norwegian Centre for Addiction Research (SERAF) in Oslo are examining sustained-release naltrexone – a non-addictive opioid antagonist that blocks the effects of opiates in the brain. Dr Mørland is hopeful that his research will make it possible to affect opiates even before they reach the brain.
An opiate roadblock
“It may be possible to block these substances from ever entering the brain, thereby modifying the effect of heroin,” Dr Mørland adds.
As part of a new project, he and his colleagues will study the effect of a 6-MAM antibody developed by a Norwegian company. The antibody binds to the 6-MAM in the blood, making the 6-MAM molecule too large to cross the blood-brain barrier.
“If we succeed in getting this antibody to work it could block much – and maybe even all – of the effect of heroin,” the researcher concludes.
Researchers Design Variant of Main Painkiller Receptor
Opioids, such as morphine, are still the most effective class of painkillers, but they come with unwanted side effects and can also be addictive and deadly at high doses. Designing new pain-killing drugs of this type involves testing them on their corresponding receptors, but access to meaningful quantities of these receptors that can work in experimental conditions has always been a limiting factor.
Now, an interdisciplinary collaboration between researchers at the University of Pennsylvania has developed a variant of the mu opioid receptor that has several advantages when it comes to experimentation. This variant can be grown in large quantities in bacteria and is also water-soluble, enabling experiments and applications that had previously been very challenging or impossible.
The study was led by Renyu Liu, an assistant professor in the Department of Anesthesiology and Critical Care at Penn’s Perelman School of Medicine, and Jeffery Saven, an associate professor in the Department of Chemistry in the School of Arts and Sciences. Jose Manuel Perez-Aguilar, then a graduate student in the Department of Chemistry, and Jin Xi, Felipe Matsunaga and Xu Cui, lab members in the Department of Anesthesiology and Critical Care, along with Bernard Selling of Impact Biologicals Inc., contributed significantly to this study.
Their research was published in the Journal PLOS ONE.
The mu opioid receptor belongs to a class of cellular membrane proteins called G protein-coupled receptors, or GPCRs. Involved in wide range of biological processes, these receptors bind to molecules in the environment, initiating cellular signaling pathways. In the case of this receptor, binding to opioid molecules leads to a profound reduction of pain but also to a variety of unpleasant and potentially fatal side-effects, a problem that researchers from multiple disciplines are attempting to address.
“There are two directions for solving this problem in basic science, either working on the opioid molecule or working on the receptor,” Liu said. “We’re doing the latter.”
Experimenting on the mu opioid receptor has been challenging for several reasons. The human receptor itself is relatively scarce, appearing in small quantities on only a few types of cells, making harvesting appreciable amounts impractical. Researchers have also been unable to grow it recombinantly — genetically engineering bacteria to express the protein en masse — as some parts of the protein are toxic to E.coli. Hydrophobic, or water-hating, amino acid groups on the exterior of the receptor that help it sit in the cell’s membrane also make it insoluble in water when isolated.
The researchers set out to address these challenges by computationally designing variants of the mu opioid receptor. This task had challenges of its own; their research was conducted long before the crystal structure of receptor was known.
“The problem with this receptor is that the native structure has only very recently been solved and only a significant re-engineered mouse model at that,” Liu said. “When we started this project, we were blind.”
Starting with only the gene sequence for the human version of the receptor, the researchers knew the order of the protein’s amino acids but not how they were folded together. The structures for other GPCRs, such as rhodopsin and the beta-2 adrenergic receptor, were known at the time, however.
“Based on the comparison of our sequence to the sequences of those GPCRs, we built a computer model of the protein,” Saven said. “When the structure of the mouse version of this receptor appeared, we were able to compare our model to that structure, and they matched up really well.”
From that comparison, the researchers were able to identify the hydrophobic amino acids on the exterior of the structure, as well as some of those that were potentially toxic to E. coli.
“The objective then was to redesign those exterior amino acids,” Saven said. “Based on the physical and chemical interactions these amino acids have with each other and with water, we were able to identify sequence combinations that are consistent with the model — where atoms don’t overlap in space — and preferentially occupy the exterior surface with ones that are water soluble.”
Replacing 53 of the protein’s 288 amino acids, the research team introduced the new gene sequence into E. coli, which were able to produce large quantities of the variant. Beyond looking like the now-available mouse mu opioid receptor, the researchers were able to show its value to future studies by performing functional tests.
“We showed that this water-soluble form of the protein can compete with the native, membrane-based form when binding with antagonists that are fluorescently labeled,” Saven said. “You can watch the fluorescence shift as more of these water-soluble variants are floating around in the solution.”
The team’s computational approach enables further iterations of the variant to be more easily designed, meaning it can be tweaked alongside experimental conditions.
“This is a great product that can do a lot of things,” Liu said. “You can use this variant to look at the structure-function relationship for the receptor, or even potentially use it as a screening tool.”
Team Points to Brain’s ‘Dark Side’ as Key to Cocaine Addiction
Scientists at The Scripps Research Institute (TSRI) have found evidence that an emotion-related brain region called the central amygdala—whose activity promotes feelings of malaise and unhappiness—plays a major role in sustaining cocaine addiction.
In experiments with rats, the TSRI researchers found signs that cocaine-induced changes in this brain system contribute to anxiety-like behavior and other unpleasant symptoms of drug withdrawal—symptoms that typically drive an addict to keep using. When the researchers blocked specific brain receptors called kappa opioid receptors in this key anxiety-mediating brain region, the rats’ signs of addiction abated.
“These receptors appear to be a good target for therapy,” said Marisa Roberto, associate professor in TSRI’s addiction research group, the Committee on the Neurobiology of Addictive Disorders. Roberto was the principal investigator for the study, which appears in the journal Biological Psychiatry.
Carrot or Stick?
In addition to its clinical implications, the finding represents an alternative to the pleasure-seeking, “positive” motivational circuitry that is traditionally emphasized in addiction.
While changes in these pleasure-seeking brain networks may dominate the early period of drug use, scientists have been finding evidence of changes in the “negative” motivational circuitry as well—changes that move a person to take a drug not for its euphoric effects but for its (temporary) alleviation of the anxiety-ridden dysphoria of drug withdrawal. George F. Koob, chair of TSRI’s Committee on the Neurobiology of Addictive Disorders, has argued that these “dark side” brain changes mark the transition to a more persistent drug dependency.
In a series of recent studies, TSRI researchers including Roberto and Koob have highlighted the role of one of these dark-side actors: the receptor for the stress hormone CRF. Found abundantly in the central amygdala, CRF receptors become persistently overactive there as drug use increases, and that overactivity helps account for the negative symptoms of drug withdrawal.
The central amygdala also contains a high concentration of a class of neurotransmitters called dynorphins, which bind to kappa opioid receptors. Much like the CRF system, the dynorphin/kappa opioid system mediates negative, dysphoric feelings—and there have been hints from previous studies that CRF doesn’t work alone in producing such feelings during addiction.
“Our hypothesis was that the dynorphin/kappa opioid receptor system in the central amygdala also becomes overactive with excessive cocaine use,” said Marsida Kallupi, first author of the paper, who was a postdoctoral research associate in Roberto’s laboratory at the time of the study.
Such overactivity would be expected to arise as the brain struggles to maintain “reward homeostasis”—a middle-of-the-road balance between pleasure and displeasure—despite frequent drug-induced swerves toward euphoria. “Dynorphin possibly acts to balance the euphoric effects produced by other opioid systems during recreational drug use,” said Scott Edwards, who is a research associate in the Koob laboratory and a co-author of the study.
Reducing Signs of Addiction
When the TSRI researchers gave rats extended access to cocaine, the rats escalated their daily intake as many human users would. Sensitive electrophysiological measurements revealed signs of a persistent functional overactivity of the GABAergic system in the rats’ central amygdalae—which corresponds to an anxiety-like state in the animals. Probing with compounds that activate or block kappa opioid receptors, the scientists found signs that these receptors, like CRF receptors, do indeed help drive the central amygdala into overactivity during excessive cocaine use.
When the researchers blocked the kappa opioid receptors, central amygdala overactivity was greatly reduced. The same kappa opioid receptor-blocking treatment (antagonist) also reduced two standard signs of addiction in cocaine-using rats—the escalating hyperactive behavior each time the drug is taken and the anxiety-like behavior during withdrawal.
These results give Roberto and her colleagues hope that a similar treatment might help human cocaine addicts feel less compelled to keep using. Kappa opioid receptor blockers are already being developed for the treatment of depression and anxiety.
Blocking negative-motivational factors such as the kappa opioid and CRF systems also has the potential advantage that it spares the positive motivational pathways—the targets of older addiction therapies such as naltrexone. “We need to keep our positive motivational pathways intact so that they can signal the many normal rewarding events in our lives,” said Roberto. By contrast, she suspects, our negative motivational pathways involving CRF and kappa opioid receptors become abnormally active only in disease states such as addiction, and thus may be blocked more safely.