New compound could supercharge naloxone in fight against opioid overdoses
New compound supercharges naloxone
STANFORD MEDICINE
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NALOXONE (ORANGE) TREATS OPIOID OVERDOSE BY KICKING OUT OPIOIDS (PINK) FROM THE OPIOID RECEPTOR (TEAL). THE NEWLY DISCOVERED COMPOUND 368 (PURPLE) STRENGTHENS THE BINDING OF NALOXONE TO THE OPIOID RECEPTOR, MAKING IT A MORE EFFECTIVE LIFE-SAVING MEDICINE.
view moreCREDIT: EMILY MOSKAL/STANFORD MEDICINE
Every great superhero needs a sidekick. Now, scientists may have found a drug-busting partner for naloxone.
Naloxone is an opioid antidote that has saved tens of thousands of lives by rapidly reversing opioid overdoses in more than 90% of cases in which it is used. But its powers are temporary, lasting only 30 to 90 minutes. The rise of potent, long-acting opioids such as fentanyl means that someone brought back from the brink can still overdose after the naloxone wears off.
In a new study, Stanford Medicine scientists and collaborators have discovered a novel compound that can work alongside naloxone, supercharging its life-saving effects.
When tested in mice, adding the compound to a miniscule dose of naloxone made it as powerful as the conventional dosage, with the added benefit of milder withdrawal symptoms.
Naloxone, which is given as a nasal spray or injection, works by seizing opioid receptors, kicking out opioids and taking their place. (Naloxone has no addictive properties of its own.) The researchers found that the new compound — known for now as compound 368 — binds next to naloxone on opioid receptors and helpfully holds naloxone in place.
The findings will be published July 3 in Nature.
“Naloxone binding to an opioid receptor turns it mostly off, but not all the way,” said Evan O’Brien, PhD, a postdoctoral scholar in molecular and cellular physiology and the lead author of the new study. “Our data shows that compound 368 is able to increase the binding of naloxone and turn the receptor off more completely.”
The senior authors of the study are Brian Kobilka, MD, professor of molecular and cellular physiology and the Hélène Irwin Fagan Chair in Cardiology at Stanford Medicine; Jay McLaughlin, PhD, professor of pharmacodynamics at the University of Florida College of Pharmacy; and Susruta Majumdar, PhD, professor of anesthesiology at the Washington University School of Medicine in St. Louis.
A new type of drug
The new compound belongs to an unusual class of drugs that don’t directly target the active site on receptors. Instead, they bind elsewhere on the receptor but trigger a structural change that alters the active site. Known as allosteric modulators (allos meaning “other” in Greek), they create new possibilities in drug development, but are trickier to identify, O’Brien said.
“Allosteric modulators are not common yet, and they’re a lot more difficult to discover and to work with,” he said.
Compound 368 is the first known allosteric modulator that can help turn off opioid receptors.
The researchers picked out compound 368 from a library of 4.5 billion compounds. Using advanced high-throughput techniques, they were able to screen the entire molecular library in just two days. To identify potential allosteric modulators that could cooperate with naloxone, they selected for compounds that bind only to receptors already saturated with naloxone.
Compound 368 — an otherwise rather unremarkable compound, O’Brien said — stood out for its ability to tightly bind to opioid receptors only in the presence of naloxone. Like a loyal sidekick, it doesn’t work with other drugs, and it doesn’t work alone.
Powers combined
When researchers exposed cells with opioid receptors to compound 368, they found that the compound alone made little difference. But when cells were given the compound with naloxone, the combination was a powerful deterrent against opioid binding.
The more compound 368 they added, the better naloxone was able to block opioids, including morphine and fentanyl.
“The compound itself doesn’t bind well without naloxone,” O’Brien said. “We think naloxone has to bind first, and then compound 368 is able to come in and cap it in place.”
Indeed, using cryoEM imaging to visualize frozen molecular structures, the researchers found that compound 368 docks right next to naloxone on the opioid receptor, forming bonds that secure the drug in place and slow its natural degradation by the body.
Boosting naloxone
Next, collaborators in McLaughlin’s lab tested the new compound in mice that had been given morphine. Because opioids reduce pain sensation, the researchers observed how quickly a mouse removed its tail from hot water. The stronger the opioid antidote, the faster a mouse would take its tail out of the water.
When mice on morphine were treated with compound 368 alone, nothing changed.
“The compound in mice, at least from the assays we’ve run, does nothing on its own,” O’Brien said. “We don’t observe any off-target effects. We don’t see anything happen to the mice even when we inject a massive amount of compound 368.”
This was exactly what the researchers had predicted from their molecular work and a good sign of the compound’s safety, he added.
When they also gave the mice a small dose of naloxone — an amount that typically would have no effect — the pairing with compound 368 dramatically improved naloxone’s effects.
“When we start to give them more and more of compound 368 with that low dose of naloxone, they take their tail out of the water pretty quickly,” O’Brien said.
Other effects of opioids, such as respiratory depression (the usual cause of death in opioid overdoses), were also reversed by a small dose of naloxone enhanced with the new compound.
Remarkably, the combination of compound 368 with a half dose of naloxone was strong enough to counter fentanyl, which is about 100 times more potent than morphine and the main culprit of overdoses in the United States.
By requiring less naloxone, the new compound could also ease the withdrawal symptoms that opioid users experience after overdose treatment. These symptoms — including body aches, shivering, nausea and diarrhea — are immediate and can be extremely uncomfortable, O’Brien said.
The researchers found that a low dose of naloxone plus compound 368 could reverse the effects of opioids with much milder withdrawal symptoms — in mice, this meant less teeth chattering, jumping and diarrhea.
Saving lives
The team, with the Majumdar lab’s expertise in medicinal chemistry, is now tweaking compound 368 so it can help naloxone counter strong opioids for longer durations.
“We’re still working on optimizing the compound’s properties for those longer-lasting effects,” O’Brien said. “But first showing that it works cooperatively with these low doses of naloxone suggests that we’re on the right track.”
O’Brien is optimistic that this track will lead to trials in humans. Overdoses from synthetic opioids, primarily fentanyl, continue to surge, killing nearly 74,000 Americans in 2022. “The more tools at our disposal, the better we’ll be able to fight this epidemic of fentanyl overdoses,” he said.
Researchers from Kurume University, SLAC National Acceleration Laboratory, Princeton University and University of Copenhagen also contributed to the work.
The study received funding from an American Diabetes Association Postdoctoral Fellowship, an American Heart Association Postdoctoral Fellowship, the National Institutes of Health (grant RO1DA057790) and the Chan Zuckerberg Biohub.
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About Stanford Medicine
Stanford Medicine is an integrated academic health system comprising the Stanford School of Medicine and adult and pediatric health care delivery systems. Together, they harness the full potential of biomedicine through collaborative research, education and clinical care for patients. For more information, please visit med.stanford.edu.
JOURNAL
Nature
METHOD OF RESEARCH
Computational simulation/modeling
SUBJECT OF RESEARCH
Animals
ARTICLE PUBLICATION DATE
3-Jul-2024
Experimental drug supercharges medicine that reverses opioid overdose
Adding newly ID’d compound makes naloxone more potent, longer lasting, mouse study shows
The ongoing opioid epidemic in the U.S. kills tens of thousands of people every year. Naloxone, sold under the brand name Narcan, has saved countless lives by reversing opioid overdoses. But new and more powerful opioids keep appearing, and first responders are finding it increasingly difficult to revive people who overdose.
Now, researchers have found an approach that could extend naloxone’s lifesaving power, even in the face of ever-more-dangerous opioids. A team of researchers from Washington University School of Medicine in St. Louis, Stanford University and the University of Florida have identified potential drugs that make naloxone more potent and longer lasting, capable of reversing the effects of opioids in mice at low doses without worsening withdrawal symptoms. The study is published July 3 in Nature.
“Naloxone is a lifesaver, but it’s not a miracle drug; it has limitations,” said co-senior author Susruta Majumdar, PhD, a professor of anesthesiology at Washington University. “Many people who overdose on opioids need more than one dose of naloxone before they are out of danger. This study is a proof of concept that we can make naloxone work better — last longer and be more potent — by giving it in combination with a molecule that influences the responses of the opioid receptor.”
Opioids such as oxycodone and fentanyl work by slipping inside a pocket on the opioid receptor, which is found primarily on neurons in the brain. The presence of opioids activates the receptor, setting off a cascade of molecular events that temporarily alters how the brain functions: reducing the perception of pain, inducing a sense of euphoria and slowing down breathing. It is this suppression of breathing that makes opioids so deadly.
The molecular compound described in the paper is a so-called negative allosteric modulator (NAM) of the opioid receptor. Allosteric modulators are a hot area of research in pharmacology, because they offer a way to influence how the body responds to drugs by fine-tuning the activity of drug receptors rather than the drugs themselves. Co-author Vipin Rangari, PhD, a postdoctoral fellow in the Majumdar lab, did the experiments to chemically characterize the compound.
Naloxone is an opioid, but unlike other opioids, its presence in the binding pocket doesn’t activate the receptor. This unique feature gives naloxone the power to reverse overdoses by displacing problematic opioids from the pocket, thereby deactivating the opioid receptor. The problem is that naloxone wears off before other opioids do. For example, naloxone works for about two hours, while fentanyl can stay in the bloodstream for eight hours. Once naloxone falls out of the binding pocket, any fentanyl molecules that are still circulating can re-attach to and re-activate the receptor, causing the overdose symptoms to return.
The research team — led by co-senior authors Majumdar; Brian K. Kobilka, PhD, a professor of molecular and cellular physiology at Stanford University; and Jay P. McLaughlin, PhD, a professor of pharmacodynamics at the University of Florida — set out to find NAMs that strengthen naloxone by helping it stay in the binding pocket longer and suppress the activation of the opioid receptor more effectively.
To do so, they screened a library of 4.5 billion molecules in the lab in search of molecules that bound to the opioid receptor with naloxone already tucked into the receptor’s pocket. Compounds representing several molecular families passed the initial screen, with one of the most promising dubbed compound 368. Further experiments in cells revealed that, in the presence of compound 368, naloxone was 7.6 times more effective at inhibiting the activation of the opioid receptor, partly because naloxone stayed in the binding pocket at least 10 times longer.
“The compound itself doesn’t bind well without naloxone,” said Evan O’Brien, PhD, the lead author on the study and a postdoctoral scholar in Kobilka’s lab at Stanford. “We think naloxone has to bind first, and then compound 368 is able to come in and cap it in place.”
Even better, compound 368 improved naloxone’s ability to counteract opioid overdoses in mice and enabled naloxone to reverse the effects of fentanyl and morphine at 1/10th the usual doses.
However, people who overdose on opioids and are revived with naloxone can experience withdrawal symptoms such as pain, chills, vomiting and irritability. In this study, while the addition of compound 368 boosted naloxone’s potency, it did not worsen the mice’s withdrawal symptoms.
“We have a long way to go, but these results are really exciting,” McLaughlin said. “Opioid withdrawal likely won’t kill you, but they’re so severe that users often resume taking opioids within a day or two to stop the symptoms. The idea that we can rescue patients from overdose with reduced withdrawal might just help a lot of people.”
Compound 368 is just one of several molecules that show potential as NAMs of the opioid receptor. The researchers have filed a patent on the NAMs, and are working on narrowing down and characterizing the most promising candidates. Majumdar estimates that it will be 10 to 15 years before a naloxone-enhancing NAM is brought to market.
“Developing a new drug is a very long process, and in the meantime new synthetic opioids are just going to keep on coming and getting more and more potent, which means more and more deadly,” Majumdar said. “Our hope is that by developing a NAM, we can preserve naloxone’s power to serve as an antidote, no matter what kind of opioids emerge in the future.”
JOURNAL
Nature
METHOD OF RESEARCH
Experimental study
SUBJECT OF RESEARCH
Animals
ARTICLE TITLE
Negative allosteric modulation of the μ-opioid receptor
ARTICLE PUBLICATION DATE
3-Jul-2024
COI STATEMENT
B.K.K. is a founder and consultant for ConfometRx. S.M. is a founder of Sparian Biosciences. E.S.O, K.K., V.A.R, S.M. and B.K.K. have filed a patent around the new NAM compound acting through μOR. The authors declare no other competing interests. B.J.K. is an employee of Lotus Separations.
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