For many people with advanced Type 2 diabetes, taking insulin is a regular part of their routine, helping them control their blood sugar by signaling the metabolism of glucose. But recently, researchers have been investigating GLP-1, a peptide that gets activated when people eat, triggering insulin through a more natural pathway.

“Proteins do a lot of the work in cells,” said E. James Petersson, an associate professor of chemistry in the University of Pennsylvania’s School of Arts and Sciences. “Peptides are shorter, and they're not really functional as machines in the same way that proteins are. But what they can do is signal molecules. One cell will secrete a peptide, and it will travel through the bloodstream and activate another type of cell.”

The problem with giving patients GLP-1 to trigger insulin production is that the peptide degrades in about two minutes due to natural enzymes in the body that break it apart, a process called proteolysis. In a paper published in the Journal of the American Chemical Society and highlighted in Nature, Penn researchers used in-vitro experiments and in-vivo studies in rats to demonstrate that, by modifying the peptide backbones, they can block interactions with the enzymes that degrade peptides and can produce a stabilized, longer-lasting version of the drug.

The research was led by Petersson; Matthew Hayes, an associate professor of nutritional neuroscience in psychiatry at Penn’s Perelman School of Medicine; Ph.D graduate Xing Cheng; postdoctoral fellow Elizabeth Mietlicki-Baase, now an assistant professor in the Department of Exercise and Nutrition Sciences in the University at Buffalo’s School of Public Health and Health Professions; and Ph.D. candidate Taylor Barrett. Petersson said that this interdisciplinary research was made possible by seed funding through the Penn Institute for Translational Medicine and Therapeutics program and by Barrett’s fellowship through the Chemistry-Biology Interface Training Grant.

“Every amino acid in a peptide is connected by an amide bond,” Petersson said. “We've been working on modifying that carbon, oxygen and nitrogen connection to a carbon, sulfur and nitrogen connection, a thioamide. We wanted to test whether this could be useful in a therapeutic context, and so we picked GLP-1 because it’s been established that if you could stabilize it then it would be a valuable diabetes treatment.”

One important property of GLP-1 is that it’s not degraded everywhere throughout the peptide; there's one specific bond that breaks apart. The researchers knew that being able to stop that cleavage would give them a much more stable version of the drug, so they replaced the oxygen atom located at that bond with a sulfur atom. With just this single atom substitution, they were able to increase the half-life of the drug from two minutes to 12 to 24 hours.

After performing in vitro tests of this method, Petersson struck up a collaboration with Hayes to see if it could actually be used in vivo. They were able to show that, in rats, the peptide was indeed longer lived and more potent than the native GLP-1. They showed that the modified peptide was as much as 750 times more stable than the natural variety, giving rats smaller blood-sugar spikes after meals.

One of the next steps, Petersson said, is to use this method to investigate other properties of GLP-1.

“In addition to triggering insulin secretion, GLP-1 actually has a whole bunch of other attributes that are beneficial for diabetes,” he said. “For instance, it reduces appetite and changes fat metabolism. This makes it a potential treatment for the obesity that's often tied to diabetes. But a lot of the reasoning for the effects on appetite, for example, aren't understood. It’s a central nervous system effect, but it's supposed to be something that's secreted in your gut, so why is it affecting how your brain works? One of the things that we want to do is use our stabilized peptide, labeled with a fluorophore, to try to understand that. Matt has already started to do some of that with fluorescent labeled peptides that aren't stabilized, so the fact that ours is longer lived will hopefully give better images of the brain.”

Although the drug market around GLP-1 is currently crowded, the researchers are also investigating other potential uses for their method of stabilizing peptides.

“We picked GLP-1 as a proof of principle because there was all this established biology and medical evidence that it was valuable,” Petersson said, “but the thioamide stabilization is a really general technology, and so we're interested in applying it to all sorts of different signaling peptides. We can use it to study how peptides are trafficked within the body and how they affect different parts of the nervous system and brain. We're also starting to collaborate with other people who are interested in the biology of signaling peptides in therapeutic and diagnostic applications.”

This new method, Petersson said, does a really nice job of stopping proteolysis while being a very small modification of the peptide. The fact that they can prevent degradation without affecting other aspects of the peptide is a key finding.

“This research shows what an amazing effect just a single atom change can have,” Petersson said. “We really need to think carefully about the chemical structure of the molecules. Understanding molecular interactions even down to single atom detail can be crucial in making valuable molecules for in-vivo studies. I think this method will allow us to learn something really interesting about fundamental biology, and our long-term plan is to apply it where all other ways to stabilize injectable peptides have failed.”

This research was supported by funding from the University of Pennsylvania, as well the National Institutes of Health; National Center for Research Resources Grant UL1RR024134; National Center for Advancing Translational Sciences grants UL1TR000003, R01 DK096139 and T32 GM071399; and National Science Foundation Grant NSF CHE-1150351.