Researchers may have solved decades-old mystery behind benzodiazepine side effects

Benzodiazepines like Valium and Xanax are often prescribed to treat anxiety, insomnia and seizures. While these drugs can be effective as a short-term treatment, researchers are trying to better understand the impact of benzodiazepines after extended use. Some experts believe long-term use of the medication may influence inflammation levels in our bodies, as previous research has shown that benzodiazepines may increase the risk of developing or worsening inflammatory conditions, like lung inflammation and inflammatory bowel disease. For years, experts have tried – without much success – to better understand the molecular mechanisms that may be driving these side effects. 

Now, a research team led by Virginia Commonwealth University and Columbia University has gained novel insights into a protein suspected to be involved in benzodiazepine-related inflammation. Their findings, published March 27 in The Proceedings of the National Academy of Sciences, could inform strategies to improve benzodiazepine drug design as well as open new opportunities for treating inflammation-related conditions, including certain cancers, arthritis, Alzheimer’s disease and multiple sclerosis.

“Numerous attempts have been made to determine the structure and elucidate the function of this mysterious membrane protein family,” said Youzhong Guo, Ph.D., an associate professor in the VCU School of Pharmacy’s Department of Medicinal Chemistry and one of the lead researchers of the new study. “Now, after decades of research, we finally have promising evidence that resolves some of the mysteries around this protein and could be crucial for advancing benzodiazepine drug design.” 

Benzodiazepines produce their therapeutic effect by binding with GABA_A receptors in the brain; however, the drug has an equally strong affinity to human mitochondrial tryptophan-rich sensory proteins (HsTSPO1), located on the outer membrane of mitochondria in cells. This type of protein is linked to several neurodegenerative diseases, including Alzheimer’s, and researchers have suspected that HsTSPO1 may be involved in certain side effects of benzodiazepine drugs.

However, both the structure and function of this protein family have been debated within the scientific community, inhibiting efforts to understand its role in disease and develop effective therapeutics. Many scientists have theorized that HsTSPO1’s potential function is transporting cholesterol across membranes to regulate the development of steroid hormones. But Guo and Wayne Hendrickson, Ph.D., a professor in the Department of Biochemistry and Molecular Biophysics at Columbia’s Vagelos College of Physicians and Surgeons and co-author of the new study, believed that HsTSPO1 is more likely to have a different function. 

“Tryptophan-rich sensory proteins like HsTSPO1 are found in all forms of life, from bacteria and plants to animals and humans,” said Guo, who also serves on research faculty at the VCU Center for Drug Discovery. “We know that this type of protein functions as enzymes in bacteria, and when you consider evolutionary theory, the same type of protein is likely to be an enzyme in humans as well.” 

Guo’s interest in this field began when he was a postdoctoral researcher at Columbia, where Hendrickson was his advisor. The two have been working together ever since to try to solve the mysteries behind this protein’s structure and function. 

HsTSPO1’s structure has remained unresolved for so long in part because of the methods used to analyze membrane proteins. The membrane of cells and organelles like mitochondria are composed of a lipid bilayer, with proteins either attached to or embedded within the structure. Researchers use detergents to extract and stabilize these proteins. However, the process can interfere with protein-lipid interactions that are often essential for the structural stability and functionality of these proteins. 

To overcome this challenge, Guo and his colleagues developed a detergent-free method, named the native cell membrane nanoparticles system, which uses membrane-active polymers to isolate and stabilize membrane proteins while maintaining their interactions with the native lipids. Using this technology, the researchers were able to study HsTSPO1 in a state that more closely reflects its natural cell membrane environment, revealing new insights into the protein’s structure and interactions with other compounds. 

“Protein instability caused by detergents had thwarted our previous efforts to fully characterize its structure and function,” Guo said. “However, in our analysis, we found that HsTSPO1 performed its function when cholesterol was present, demonstrating how crucial it is to study this protein in an environment that is similar to its natural habitat. Similar to if you take a fish out of the water, it’s still a fish, but it will behave very differently.”

Through this method, the research team found evidence to suggest that HsTSPO1 functions as an enzyme. They discovered that HsTSPO1 breaks down protoporphyrin IX, a compound found in oxygen-rich red blood cells, to create a novel product that the scientists have named bilindigin. This product helps control the level of “reactive oxygen species” (ROS) in our bodies, a type of compound that can lead to inflammation and kill cells if left unregulated. This finding suggests that, when valium and other benzodiazepines bind to HsTSPO1, they inhibit the protein’s ability to manage ROS levels in our cells. This may help explain why such medications cause side effects over time, though more research is needed to fully understand whether these molecular mechanisms play a part in driving adverse side effects.

“The enzyme activity that we found for HsTSPO1 both reduces the production and the neutralization of ROS,” Hendrickson said. “This discovery then provides a rationale for fresh approaches in drug discovery.” 

The new insights into HsTSPO1’s function could help pharmaceutical companies develop improved benzodiazepines. Furthermore, because of its newly discovered role in regulating reactive oxygen species, the researchers say HsTSPO1 might serve as a promising drug target for monitoring and treating neurodegenerative diseases, like Alzheimer’s, as well as other inflammation-related conditions that have connections to HsTSPO1. This includes some cancers, arthritis and MS. 

“Benzodiazepines are still widely used to treat anxiety, insomnia, seizures and other conditions. Now that we have an understanding of how HsTSPO1 works, we could potentially create better drugs with less side effects,” Guo said. “But on a larger scale, our insights into this protein could have significant implications for developing new therapeutic options for patients impacted by inflammatory diseases.”