Opioid Candidate Suppresses Pain in Mice with Fewer Side Effects

Opioid medications offer people relief from debilitating pain, but these drugs are associated with the risk for addiction, miserable withdrawal symptoms and the potential for fatal overdose. Researchers headed by teams at the Washington University School of Medicine, St. Louis, at the University of Florida, and at the Stanford University School of Medicine have identified a strategy to design safer opioids. They showed that an experimental opioid, which binds to an unconventional spot on the receptor, suppresses pain in animal models with fewer side effects—most notably those linked to fatal overdoses.

Research co-lead Susruta Majumdar, PhD, at the University of Health Sciences and Pharmacy at St. Louis and the Washington University School of Medicine, St. Louis, and colleagues reported on their development in ACS Central Science, in a paper titled “Signaling Modulation Mediated by Ligand Water Interactions with the Sodium Site at μOR.”

Opioid medications tap into the body’s natural system for mitigating pain by activating pain-suppressing opioid receptors on nerve cells in the brain. “The mu opioid receptor (μOR) is a target for clinically used analgesics,” the team wrote. Although such drugs are intended to help people, they can have adverse effects. People who take opioids may become physically dependent on them, leading, on cessation, to withdrawal symptoms such as muscle pain, nausea and vomiting. In addition, opioids make breathing slow and shallow, a side effect that can become fatal. The authors cited figures indicating that opioid overdose was responsible for more than 82,820 deaths in 2023 in the U.S. alone. As they noted, “… developing newer and safer analgesics targeting the μOR is an urgent necessity.”

Attempts to design safer opioids have largely focused on identifying molecules that bind to the same spot on the receptor, known as the active site, where the body’s own pain-relieving signals attach. “Most of these approaches have targeted the orthosteric binding site except for a few reported allosteric modulators,” the investigators continued.

In an earlier study, researchers found a molecule called C6 guano, which can activate the opioid receptor when it binds outside the active site. C6 guano interacts with a location inside the opioid receptor that typically responds to sodium ions. “Recently we reported a novel strategy to design functionally selective opioids by targeting the sodium binding allosteric site in μOR with a supraspinally active analgesic named C6guano,” the scientists noted. Despite its promising effects, C6 guano has a significant drawback, in that it cannot pass through the blood-brain barrier. The team led by Majumdar, together with Jay McLaughlin, PhD, at the University of Florida Gainesville, Haoqing Wang, PhD, and Ruth Hüttenhain, PhD, at the Stanford University School of Medicine, set out to improve on this discovery by identifying a similar molecule with the potential to travel from the blood stream to opioid receptors in the brain.

To find an alternative that also binds to the opioid receptor, the researchers synthesized and evaluated 10 compounds with chemistry that might enable passage through the blood-brain barrier. During their initial testing in cells, they identified the most promising candidate derived from fentanyl, called RO76. “The design of analogs based on fentanyl templates was aimed to target the water molecules present in the Na+ binding site,” the team stated. “We synthesized 10 analogs and studied their pharmacology using cell based in vitro assays … Synthesis and screening of a small library of analogs led to the identification of a lead named RO76.”

By capturing molecules in proximity of the activated receptor, the team showed that RO76 produces a signal within cells that differs from those initiated by classic opioids, such as morphine. “A cryoEM structure of RO76 bound to the μOR-Gi complex confirmed that RO76 interacts with the sodium site residues through a water molecule, unlike C6guano which engages the sodium site directly,” they further explained.

The investigators then evaluated the compound’s efficacy in mice when injected subcutaneously (sc) or given orally. They first determined that the compound could penetrate into the brain. “In mice, RO76 showed optimal brain penetration (brain: plasma ratio = 0.5) compared to C6guano and was active when administered by both the subcutaneous and oral routes, reaffirming our design strategy,” they noted. The mouse experiments also indicated that RO76 appeared to suppress pain as effectively as morphine. But when comparing the opioids’ effects on the animals breathing rates, they found that RO76 slowed breathing far less. Likewise, when they gave the mice an opioid-blocking medication, the mice chronically taking RO76 experienced fewer withdrawal symptoms than those taking morphine. “RO76 did not decrease breath rate at doses of 10 or 30 mg/kg, sc, and in general had reduced respiratory depression and physical dependence after chronic administration as compared to morphine in mice,” they reported. “Thus, targeting the Na+ site indirectly through the bitopic approach with water molecules leads to a systemically active and a safer μ opioid antinociceptive agent.”

The studies in addition indicated that, when given by mouth, the new fentanyl derivative had similar, if slightly lower, pain-suppressing effects than when injected under the skin of the animals.

Noting limitations of their approach, the researchers say that their results suggest RO76 has the potential for development as an oral medication for humans, and indicate that the strategy may have wider utility. “Collectively, the behavioral studies demonstrate that RO76 is a μOR-selective agonist displaying similar antinociception to morphine, but with reduced adverse effects of respiratory depression or symptoms of withdrawal denoting reduced physical dependence … we show that targeting water molecules in the sodium binding pocket may be an avenue to modulate signaling properties of opioids, and which may potentially be extended to other G-protein coupled receptors where this site is conserved.”