Opioids are the gold standard of chronic pain treatment, but they come with a host of negative side effects including constipation, addiction potential and respiratory depression that can lead to death. This study, published in Scientific Reports, offers a potential new way to treat acute and chronic pain by reducing the amount of opioid needed for pain relief while also lowering its addiction potential.

“We have been investigating the role of Heat shock protein 90 in regulating opioid signaling in the spinal cord for some time,” said John Streicher, PhD, member of the Comprehensive Center for Pain & Addiction at UArizona Health Sciences and a professor in the College of Medicine — Tucson’s Department of Pharmacology. “This study provides proof of principle that Hsp90 isoform inhibitors are effective at improving opioid pain relief and reducing side effects. This is the critical link that makes our work translationally relevant, giving us a clear path forward to develop a new drug that could benefit millions of people who live with chronic pain.”

Heat shock protein 90 is a chaperone protein that helps other proteins function, including those that promote tumor growth. It has been studied primarily in the context of cancer. Streicher is leading in a long-term effort to investigate its role in opioid receptor activation and pain relief.

Streicher’s prior research showed that Heat shock protein 90 acted upon opioid receptors in the brain differently than in the spinal cord. Inhibiting Hsp90 in the brain blocked the analgesic properties of morphine, meaning the opioid lost its ability to reduce the sensation of pain. But inhibiting Hsp90 in the spinal cord amplified the pain-relieving effects of morphine.

Building on that research, the team tested nonselective Hsp90 inhibitors in mouse models and saw a twofold-to-fourfold increase in the potency of pain relief provided by morphine. At the same time, tolerance was reduced and established tolerance was reversed. Tolerance is a condition where the body gets used to a medication so that more medication or a different medication is needed to achieve the same response.

Early cancer-focused studies, however, found that nonselective Hsp90 inhibitors can cause serious side effects, including macular degeneration. Streicher’s solution was to target individual isoforms of Hsp90, of which there are four.

“Isoforms are different versions of the same thing, like trim packages on a car,” Streicher said. “They are all slightly different and have similar roles, but not identical roles. So these four Hsp90 isoforms are four proteins that we can target individually.”

By using selective inhibitors to target each isoform, they were able to identify and isolate the isoforms that are active in the spinal cord from Hsp90-alpha, the one that is active in the brain. Recent reports have linked Hsp90-alpha with the serious side effect of retinal degeneration.

“We took isoform-selected inhibitors that we got from our collaborator, Brian Blagg, PhD, at the University of Notre Dame, and gave them to mice systemically via IV injection,” Streicher said. “We found that you can give these isoform-selective inhibitors by a translatable route and get the benefits. Pain relief goes up and side effects go down, and presumably we’re going to avoid some of those nasty side effects of the nonselective Hsp90 inhibitors.”

The findings suggest that selective Hsp90 inhibitors could be used as part of a dose-reduction plan in conjunction with opioid therapy prescribed by a physician for chronic pain. The goal is for doctors to be able to prescribe lower amounts of opioids that provide patients with the same pain-relieving benefits and fewer negative side effects.

“What I’m envisioning is you’d be given a pill that is a combination therapy of an opioid with one of these isoform inhibitors,” Streicher said. “The addition of that Hsp90 inhibitor would make the opioid better — it would increase the effectiveness of the pain relief and decrease the side effects.”

Streicher and his team are working to optimize the selective Hsp90 inhibitors to produce a stable drug that can be taken orally.

“Dr. Streicher’s research is an excellent example of the innovative, translational science that is needed to transform health care for pain and addiction,” said Todd Vanderah, PhD, director of the Comprehensive Center for Pain & Addiction, Regents Professor and head of the Department of Pharmacology. “This study is an important step toward developing a novel evidence-based therapy that will provide better treatment options with fewer disruptive side effects, empowering people with chronic pain to thrive.”

The research team included four co-first authors: David Duron, PhD, JD, former doctoral candidate in the Streicher Lab; Parthasaradhireddy Tanguturi, PhD, researcher/scientist in the Department of Pharmacology; former doctoral candidate Christopher Campbell, PhD; and Kerry Chou, former undergraduate student at the UArizona College of Nursing. Other co-authors included Paul Bejarano, and former doctoral candidates Katherin Gabriel, PhD, and Jessica Bowden, DVM, PhD, all of the Department of Pharmacology; Sanket Mishra, PhD, Christopher Brackett, PhD, and Brian Blagg, PhD, of the Department of Chemistry and Biochemistry at the University of Notre Dame’s College of Science; and Deborah Barlow and Karen Houseknecht, PhD, of the Department of Biomedical Sciences at the University of New England’s College of Osteopathic Medicine.

Bacteria have developed special defense mechanisms to protect themselves against viruses, which by no means infect only humans. As part of these so-called CRISPR-Cas systems, a CRISPR ribonucleic acid (crRNA), which serves as a “guide RNA,” recognizes regions of a foreign genome, such as viral DNA. The CRISPR-associated (Cas) nuclease, directed by a crRNA, then renders it harmless by cutting it like a pair of scissors. Humans have exploited this strategy: “CRISPR, often referred to as ‘gene scissors’, is the basis of many molecular technologies,” says Chase Beisel, head of the RNA Synthetic Biology department at the Helmholtz Institute for RNA-based Infection Research (HIRI) in Würzburg. The institute is a site of the Braunschweig Helmholtz Centre for Infection Research (HZI) in cooperation with the Julius-Maximilians-Universität (JMU) of Würzburg, where Beisel holds a professorship.

The diagnostic platform LEOPARD, developed by Beisel’s lab in cooperation with JMU in 2021, also leverages CRISPR as a technology. LEOPARD has the potential to detect a variety of disease-related biomarkers in just one test. The approach is based on reprogramming RNA factors, so-called tracrRNAs. Those RNAs are naturally involved in helping produce guide RNAs used by Cas9 and different Cas12 nucleases. “LEOPARD focused on Cas9. However, CRISPR-Cas systems also include another diverse set of nucleases, called Cas12,” explains Beisel. While both Cas9 and Cas12 cut DNA targets, Cas12 can increase the output signal by performing cuts on “collateral” DNA. This can make detection technologies more sensitive and, therefore, more efficient.

The team led by Chase Beisel has now extended the unique features of LEOPARD to Cas12. The researchers have named the resulting method PUMA (Programmable tracrRNAs Unlock protospacer-adjacent Motif-independent detection of ribonucleic Acids by Cas12 nucleases). The details of their findings are the subject of a paper in the journal Nature Communications.

Overcoming hurdles

Although Cas12 nucleases are widely used in molecular diagnostics, two major limitations have persisted: Cas12-based technologies have been restricted to DNA targets, and a specific recognition sequence called a PAM, short for protospacer-adjacent motif, is required to identify the target molecule.

PUMA elegantly addresses these challenges. Like LEOPARD, this new method also relies on tracrRNAs. “Using PUMA, we can reprogram the tracrRNAs. This allows us to decide which RNA biomarker becomes a guide RNA. This guide RNA, in turn, directs Cas12 to a DNA molecule that we provide and activates the gene scissors,” explains the study’s first author, Chunlei Jiao. Chunlei Jiao, a former graduate student and postdoctoral researcher in the Beisel lab, was also involved in the development of LEOPARD. He recently started a professorship at the National University of Singapore. “DNA cutting then tells us which biomarker was present in the sample, such as biomarkers specific to different pathogens,” adds Beisel.

The novel method therefore enables the detection of RNA biomarkers using CRISPR nucleases that can normally only recognize DNA. “This is particularly important for molecular biomarkers that can only be found at the RNA level. This includes RNA viruses, for example,” says Beisel. And yet, PUMA does not require a specific recognition sequence: The PAM is contained in the DNA target molecule provided. Since the researchers provide the target molecule, they can also introduce truncated DNA. As a result, they were able to significantly increase the speed of the method.

Several birds, one stone

“PUMA has the potential to become a flexible and precise tool for RNA detection,” concludes Beisel. Finally, the team demonstrated the potential of the method by identifying five bacterial pathogens associated with acute sepsis. Their detection relied on a single universal, reprogrammed tracrRNA, which provides a simplified means of differentiating between various types of bacteria. This opens up a wide range of potential applications in medicine: “The new technology represents a novel form of CRISPR diagnostics that enables reliable molecular testing at the point of care — whether for the identification of viral or bacterial pathogens or the detection of cancer biomarkers,” says Jiao.

The research team is already planning its next steps: “Our goal is to achieve a multiplexed readout similar to that of LEOPARD and to expand the range of applications for the technology,” says Beisel, who also anticipates broad use in the research community: “We hope that our study will spur further exploration of tracrRNA reprogramming.”

In a world first study, published in BMJ Open Sport & Exercise Medicine and funded by the Health Research Council, participants completed two four-hour evening intervention sessions of prolonged sitting, and sitting interrupted with three-minute activity breaks every half hour.

The researchers found that after the participants completed the activity breaks intervention they slept for 30 minutes longer.

Lead author Jennifer Gale, PhD candidate in the Department of Human Nutrition, says sitting for long periods is associated with an increased risk of diabetes, cardiovascular disease and death.

“We know that for many of us, our longest period of uninterrupted sitting happens at home in the evening. In our previous studies we have found that getting up and doing 2-3 minutes of exercise every 30 minutes reduces the amount of sugar and fat in your blood stream after a meal.

“However, many sleep guidelines tell us we shouldn’t do longer bouts or higher intensity exercise in the hours before sleep, so we wanted to know what would happen if you did very short bouts of light intensity activity repeatedly throughout the evening,” she says.

Primary investigator Dr Meredith Peddie, Senior Lecturer in the Department of Human Nutrition, says the exercise intervention involved three exercises — chair squats, calf raises, and standing knee raises with straight leg hip extensions.

“These simple, bodyweight exercises were chosen because they don’t require equipment, or a lot of space and you can do them without interrupting the TV show you are watching.

“From what we know from other studies, you could probably get a similar effect if you walked around your house, marched on the spot, or even danced in your living room — the most important thing is that you get out of your chair regularly and move your body,” she says.

The fact this exercise resulted in longer sleep is important because insufficient sleep can negatively affect diet and has been associated with heart disease and type 2 diabetes.

“We know higher levels of physical activity during the day promotes better sleep, but current sleep recommendations discourage high-intensity exercise before bed because it can increase body temperature and heart rate resulting in poor sleep quality.

“It might be time to review these guidelines as our study has shown regularly interrupting long periods of sitting is a promising health intervention,” Dr Peddie says.

The study, appearing today in the Journal of Clinical Investigation, focused on two proteins called melanocortin 3 and melanocortin 4 found primarily on the surface of neurons in the brain that play a central role in regulating feeding behavior and maintaining the body’s energy balance.

Melanocortin 3 and melanocortin 4 impact everything from sensing long-term energy stores to processing signals from the gut regarding short-term fullness, or satiety, said U-M physiologist Roger Cone, who led the study.

The class of drugs known as GLP-1 agonists, which includes semaglutides (e.g., Ozempic) and tirzepatides (e.g., Mounjaro), have received substantial attention recently for their effectiveness in treating not only type 2 diabetes, but also obesity, heart disease and potentially addiction. They work by mimicking a natural hormone that the gut produces when it is full, triggering the brain to reduce feeding behavior.

“So the obvious question for us was: How do these GLP-1 drugs, which work by manipulating satiety signals, function when we prime the melanocortin system?” said Cone, professor of molecular and integrative physiology at the U-M Medical School and director of the U-M Life Sciences Institute where his lab is located.

Working in mouse models, Cone and his colleagues tested the effects of several hormones that reduce food intake. They compared the results in normal mice with mice that genetically lacked the MC3R protein, in mice that were given chemicals to block the activity of MC3R, and in mice that were given a drug to increase the activity of MC4R. (Because MC3R is a natural negative regulator of MC4R, meaning it decreases the activity of MC4R, blocking MC3R and increasing MC4R activity has similar effects.)

In all cases, Naima Dahir, first author of the study and a postdoctoral research fellow in Cone’s lab, and colleagues found that adjusting the melanocortin system — either by inhibiting MC3R or increasing MC4R activity — made the mice more sensitive to GLP-1 drugs and other hormones that affect feeding behavior. The mice that were given a GLP-1 drug in combination with an MC4R agonist or MC3R antagonist showed up to five times more weight loss and reduced feeding than mice receiving only the GLP-1 drugs.

“We found that activating the central melanocortin system hypersensitizes animals to the effects of not just GLP-1s, but to every anti-feeding hormone we tested,” Cone said.

The researchers also measured activity in parts of the brain thought to trigger nausea in response to GLP-1 drugs and observed no increased activation when GLP-1 drugs were combined with alterations to the melanocortin system. In contrast, priming of the melanocortin neurons significantly increased GLP-1 drug activation of neurons in hypothalamic feeding centers in the brain.

The findings indicate that pairing the existing GLP-1 drugs with an MC4R agonist could increase sensitivity to the desired effects of the drugs by up to fivefold, without increasing unwanted side effects.Ultimately, this approach could enable patients who are sensitive to the side effects to take a lower dose, or could improve the results in patients who have not responded to the existing drug dosages. Further drug development and clinical testing are needed before this can occur.

While this research has been conducted only in mouse models, Cone is optimistic that the results will translate well to humans.

“The melanocortin system is highly conserved in humans,” he said. “Everything we’ve observed in the mouse over the past decades studying these proteins has also been found in humans, so I suspect that these results would also be translatable to patients.”

This research was funded by the National Institutes of Health and Courage Therapeutics.