Three Million to One, Homing in on a Winner with Few Side Effects
Breathing slows, oxygen levels plummet and, all too often, lungs cease to function at all. An opioid again takes a life, and another person is lost to an overdose. Deaths from opioids are happening with increasing frequency throughout the world.
Since long before Dorothy fell asleep in a field of poppies alongside the Yellow Brick Road while off to see The Wizard of Oz, the powers of poppies have been legend. The sleep-inducing and pain-reducing properties stem from the natural opiates found in the seed pods of the opium poppy. Too often, morphine prescribed to control pain leads to an out-of-control addiction and, in the most serious situations, a fatal overdose.
Researchers have sought out alternatives to morphine, codeine and other opiates, options that might effectively control pain without the risk of addiction and respiratory distress. Recently, teams from the United States and Germany announced that a search built around the structure of the opioid receptor yielded a promising replacement candidate. They described their process and findings in, “Structure-based discovery of opioid analgesics with reduced side effects,” in Nature.
Planning a New Scaffold
When morphine reaches the mu-opioid receptor (μOR), it combines with two signaling proteins. One, beta-arrestin, is linked to troublesome and potentially dangerous opioid side effects, such as addiction and respiratory failure. The other, G-protein, is linked to pain relief. To reduce risks of addiction and respiratory failure, some research has focused on altering morphine, aiming to change it just enough to avoid the risks while retaining the benefits.
Aashish Manglik, MD, PhD, Stanford University School of Medicine, and his team explored a fundamentally different approach. They weren’t looking for a compound like morphine. In fact, they wanted a novel molecule constructed on a new scaffold, one dissimilar in structure to opioids but that nonetheless would fit into the stereotactic pocket of the mu-opioid receptor.
As in virtually all research, the team’s current study builds on previous findings, in this case, their own. In 2012, they identified the crystal structure of several opioid receptors. Based on those structures, the researchers created a computational screening program to find non-opioid molecules that would dock to — in essence “fit” into —μOR in a biased way, setting in motion the pathway for pain control but not the pathway linked to dangerous side effects. That program screened 3 million virtual molecules to illustrate how each might bind to the docking site of the μOR. Based on computer analysis and direct observation, the researchers winnowed down millions of possibilities to the most promising candidate, which they optimized to construct PZM21. Then they moved from computer models and bench results to in vivo tests.
Of Mice and Monitoring
The researchers employed several key tests to gather information on pain relief, respiratory depression and indicators of addiction.
One test demonstrated that mice who received PZM21 didn’t exhibit signs of pain when exposed to heat. In fact, the pain protection from PZM21 was just about as strong as that of morphine. PZM21 also kicked in more quickly and lasted longer.
In another experiment, the mice on PZM21 flicked their tails as frequently as if they hadn’t received any medication. Importantly, this showed that, although PZM21 alleviated pain, it did not dull spinal responses, an encouraging sign that PZM21 would not fatally depress respiration. Direct measurements of breathing confirmed that respiration remained close to normal on PZM21 in stark contrast to the radical respiratory depression in mice on morphine.
Finally, although there are inherent limits to watching the activity of mice to predict the behavior of people, the rodent response offered reason for optimism. Mice did not demonstrate the classic signs of addiction, drug-seeking behavior and hyperactivity. They moved around a normal amount and didn’t show a preference for PZM21 solution over saline. They weren’t revved up in search of their next high.
Complex Analyses Using Complementary Technologies
The rigorous evaluation of compounds throughout the study required the complementary abilities of a collection of spectroscopy techniques. For high-resolution electrospray ionization mass spectrometry, the scientists used either a Bruker microTOF II or a Bruker maXis MS, a quadrupole-time-of-flight machine. To determine mass, the team used the Bruker Esquire 2000 ion-trap mass spectrometer or a Bruker amaZon SL mass spectrometer. The researchers recorded 1H and 13C spectra on a Bruker Avance 360 or 600 NMR-Spectrometer.
Shaping What’s Next
Along with the importance of PZM21 as a potential substitute for dangerous opioid pain relievers, its very structure might reveal new information about the mechanisms of opioid receptors and signaling systems.
The methods used in this experiment — from virtual screening and docking to optimizing and testing — can serve as a template for employing a structure-based approach to developing other molecules that are a good fit for any number of important receptors.
Getting PZM21 from the research lab to the prescription pad will require extensive clinical trials. In the meantime, Manglik and his colleagues believe what they learned about the mu-opioid receptor specifically and structure-based methods, in general, will spark research into receptors critical to other treatments and diseases.
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