Vitamin B12 is essential to human life, and it’s intrigued Robert Doyle since his undergraduate studies at Trinity College, Dublin, Ireland. He was fascinated by the role of metal ions in medicine and biology—and vitamin B12 has a unique structure that features a cobalt-to-carbon bond. “Vitamin B12 is a very unusual metal-based system, and that bond allows B12 to do some really spectacular chemistry,” says Doyle, the Laura J. and L. Douglas Meredith Professor in the Department of Chemistry in the College of Arts and Sciences at Syracuse University and an adjunct associate professor of medicine at SUNY Upstate Medical University . “If this bond wasn’t there, this chemistry wouldn’t happen, and we wouldn’t be here.”
At work in our bodies, vitamin B12 keeps our cells and nervous system functioning properly, which includes contributing to the formation of red blood cells, the production of proteins and the replication and repair of DNA. But since our bodies don’t produce it, we acquire it mainly through the consumption of animal products, fortified foods or supplements in our diet.
For Doyle, the vitamin has been instrumental in propelling forward his research on type 2 diabetes and other diseases. One reason for that is the vitamin’s dietary pathway—and all the possibilities it presents for exploration. “There’s a unique pathway that’s designed to protect it, carry it, transport it, place it where it needs to go, keep it safe, store it,” says Doyle, who teaches the upper-level course Metals in Medicine. “There are different proteins and receptors that all play a role in protecting it—from your saliva all the way to your brain and everything in between. Because all proliferating cells need vitamin B12, if you look at it from the sense of a medicinal chemist, you realize maybe that pathway could be exploited, and so that’s where I’ve been focusing my research for the last 15 years.”
A native of Dublin, Doyle earned a doctorate in chemistry at the University of Dublin and held postdoctoral fellowships at the Australia National University, where he studied drug delivery, and Yale University, where he focused on molecular biology and protein biochemistry. He joined Syracuse’s chemistry faculty in 2005 and has built an impressive body of research in medicinal chemistry, supported by millions of dollars in grants from the National Institutes of Health (NIH), the Department of Defense (DoD) and corporate, society and foundation partners. Doyle has invented several processes related to vitamin B12 delivery that have earned patents for the University, and he’s expanded his research network to push the science forward, sharing knowledge and drawing on the expertise of collaborators with related specialties around the world. Along the way, he’s lectured at international conferences and other universities, and he’s mentored numerous students who’ve gone on to make their own scientific contributions. He also possesses an entrepreneurial spirit, cofounding Cantius Therapeutics with two collaborators from the University of Pennsylvania—Matthew Hayes, associate professor of nutritional neuroscience in psychiatry at the Perelman School of Medicine, and Bart De Jonghe, associate professor of nursing in the Department of Biobehavioral Health Sciences.
Exploiting the Vitamin B12 Pathway
One of Doyle’s specialties is a molecule coupling technique called bioconjugation. Through this process, therapeutic peptides are attached to the workhorse B12 molecule and take advantage of its transport system, journeying along the pathway to designated locations where they’re put to work. The researchers must also ensure that the attached peptides don’t interfere with B12’s functions. In Doyle’s diabetes research, receptors in the pancreas are one of the targeted areas. The pancreas produces the hormone insulin, which transports glucose throughout the body, providing energy for life processes to the cells. Type 2 diabetes—the most common form that affects 95 percent of people with diabetes—occurs when cells in the pancreas are damaged or destroyed, disrupting insulin production and causing high levels of glucose to build up in the bloodstream.
Most recently, with grants from the NIH, Doyle and his research colleagues developed a new drug lead that will help people with diabetes control their blood sugar while avoiding the common side effects of nausea, vomiting and weight loss associated with diabetes medications. This could be especially beneficial to people with diabetes who may also have cystic fibrosis, chronic obstructive pulmonary disease, sarcopenia, HIV or other diseases that require maintaining a stable weight. In another project, Doyle is using a $3 million DoD grant to pursue research that simultaneously treats chronic diabetes and obesity—a personal health issue that affects nearly 25 percent of military veterans who receive care at VA medical centers. “My group is pushing to expand the treatment of diabetes with obesity and then, separately, to treat diabetes without affecting nutritional status,” he says.
According to Doyle, the driving force in the field has been glucagon-like peptide-1 (GLP-1) receptor agonists, a group of therapeutics that spark physiological responses when combined with a GLP-1 receptor. “They have been the darlings of the diabetes pharmacopeia now for over a decade,” he says of the multibillion-dollar industry. “They have been a great success in both generating a metabolic norm and in facilitating weight loss, which has been of huge benefit to patients who are both obese and type 2 diabetic.” By targeting GLP-1 receptors in the pancreas and brain, these agonists influence insulin production and appetite control, respectively.
Strangely enough, the scientific community can credit the Gila monster—one of the only venomous lizards on the planet—with advancing the research. As Doyle explains, the reptile’s saliva contains a peptide sequence called exendin-4 that is similar to the human GLP-1 peptide, and it’s proved hugely beneficial. Now synthetically manufactured as exenatide, it’s a popular, injectable blood glucose-lowering medication. “The sequence difference in the Gila monster’s peptide meant that when you put it in a human, it stayed in us for hours, as opposed to just minutes like the human version does,” Doyle says. “If you just put our own GLP-1 in, it’s rapidly degraded and done. If you put this one in, it lasts for hours, which gave that big improvement in long-term glucose regulation.”
But, like many medications, exenatide has drawbacks. “It also had the weird effect of triggering a receptor of the brain that facilitates weight loss,” says Doyle, noting nausea and vomiting as accompanying side effects. This basically made it unusable for people with diabetes who have other diseases like cancer or cystic fibrosis and endure bouts with extreme nausea and need to maintain their weight. “We wanted to take the best part of diabetes control in these GLP-1 receptor agonists and stop all of the weight loss, nausea and vomiting,” he says. “The way to do that was to prevent it from getting to the brain.”
The solution: Doyle’s team, including Penn collaborators Hayes and De Jonghe, created a technique to produce a new GLP-1 receptor agonist. They attached exendin-4 (Ex4) to a fragment of the B12 molecule known as dicyanocobinamide (Cbi), which has no known human biological function and doesn’t penetrate the hindbrain, where illness effects are triggered. Doyle named the technique “corrination”—which recognizes B12’s central structure (the corrin ring system) and is also a play on “coronation.” For a model system, they tested the new agonist, called Cbi-Ex4, on the musk shrew, known for its ability to vomit. The results demonstrated the agonist’s proficiency at improving hyperglycemia while reducing ill effects compared to exendin-4. The findings were published in a leading journal, Cell Reports, and the team looks to continue developing the drug through a preclinical phase and into human studies. “It turns out that by placing this little corrin ring onto the peptide, it allows access to the pancreas, but has now successfully prevented access to the part of the brain where all this action comes from,” Doyle says.
Piecing Together a Super Peptide
Exendin-4 is also a main component in a “super peptide” that the Doyle group created from scratch to serve as a GLP-1 receptor agonist. Known as GEP-44, it features glucagon, exendin-4 and a gut peptide hormone that acts as an appetite suppressant. Working with longtime collaborator Christian Roth, a pediatric endocrinologist at the Seattle Children’s Hospital, the team pieced together the super peptide, which coincidentally features 44 amino acids—possibly a good omen for Syracuse, Doyle points out. “The peptides all work to do many good things,” Doyle says. “They cause weight loss and facilitate glucose regulation, but also we wanted to build in no side effects—no nausea, no vomiting—and protection of the pancreas from the inflammatory damage that diabetes causes. We were really trying to go for a tetra-fecta.”
The DoD-funded project supports research through design to clinical development. The peptide drug is also viewed as dual-use technology, so with a positive outcome it would be available to anyone. The funding allowed Doyle to acquire a peptide synthesizer for his lab—creating the opportunity to sidestep the cost and time of commercial lab work. One of the benefits of working in Doyle’s lab is that they produce the compounds in house and then screen them for their effectiveness. “It’s awesome to be able to synthesize and build your own compounds and then directly study them in a cellular model,” says Brandon Milliken, a doctoral candidate in his fifth year with Doyle’s research group.
At Work in the Lab
For Doyle, the most rewarding part of his work is mentoring students. “I sort of sow the seeds and pay the bills like a good parent!” he says. “Then, after that, you watch them take over a project and own it. I like that the most—and my favorite day is the day they get their Ph.Ds.”
Doyle, for example, proudly notes that his first three doctoral students at the University are now professors themselves, conducting research and teaching: Nerissa Viola G’09 is an associate professor in the cancer biology program at the Karmanos Cancer Institute, Wayne State University; Oluwatayo Ikotun G’09 is an assistant professor of molecular and medical pharmacology at UCLA; and Amanda Petrus G’09 is an assistant professor of chemistry at the Community College of Rhode Island.
Doyle’s group currently consists of a 10-member research team that is a mix of undergraduates and graduate students. Doctoral candidates Milliken and Ian Tinsley both enjoy Doyle’s approach to research and have co-authored research papers with Doyle for peer-reviewed journals. “Within the Doyle group, you really are hands-on learning every aspect of the project, from the rational design and drug development, to the preclinical screening and push to the clinical aspect,” Milliken says. “You’re really covering that full spectrum of drug development.”
Milliken says it’s been his dream to earn a Ph.D. at Syracuse University. He grew up on Syracuse’s Southside and graduated from Corcoran High School, where his chemistry teacher, Mrs. Day, inspired him to pursue a science career. “Science was a passion of hers and a passion of mine,” he says. “I’ll never forget talking science with her and the direction she pushed me in.”
Milliken was an undergraduate research assistant at SUNY Cortland and earned a master’s degree from Rochester Institute of Technology, where he conducted research and served as a teaching assistant. He worked as a long-term substitute teacher at Corcoran, and for SGS Galson, a Syracuse-based industrial hygiene analysis and monitoring company, before deciding to focus on a Ph.D. specializing in biochemistry and medicinal chemistry. “Professor Doyle was my top choice to work with,” he says. “His personality and character were a dead sell. He’s a great person and the research really hooked me.”
Milliken loves creating compounds and working on them through the various stages of development, he says. One of his projects involves studying the anti-cancer drug Fenretinide, and his main work has focused on developing GEP44. With this focus on the next generation of diabetes and obesity drugs, he says one of the issues with current drugs targeting weight loss is that they only show a short-term loss of 5 to 10 percent. “In some of our studies, we’ve had prolonged weight loss of upwards of 15 percent or more without adverse side effects observed with current drugs on the market, which is unheard of in the field,” he says.
Among his experiences, he journeyed to the Seattle Children’s Hospital to screen some of the drugs there with their collaborators in the Roth Laboratory. “You’re making a therapeutic and want to see it function the way that you hope,” says Milliken, who plans to work in the pharmaceutical industry doing drug design and development. “It’s nice when you’re evaluating your own design therapeutic and seeing it work in real time, in the functional way that you’ve designed it to. It’s a special feeling.”
It’s nice when you’re evaluating your own design therapeutic and seeing it work in real time, in the functional way that you’ve designed it to. It’s a special feeling.—Brandon Milliken
Tinsley is a native of central Illinois whose interest in medicinal chemistry led him to Doyle’s lab, which caught his attention because of the work on the vitamin B12 pathway for drug delivery. Like Milliken, Tinsley enjoys Doyle’s personable, often humorous, interactions with team members, and also appreciates that he gets down to business, treats them as peers and encourages scientific exploration. Tinsley majored in biology and chemistry at University of Illinois Springfield and cites a summer research experience for igniting his interest. He built on his research skills at Eastern Illinois University, where he earned a master’s degree and discovered that he enjoyed medicinal chemistry. “I’ve always had a curiosity in how drugs are made,” he says. “Can I design new drugs or a new pharmaceutical? Can I deliver it? Can I take a drug that’s OK and make it better, or could we take a drug that’s really good but has terrible side effects that we could mitigate? Rob’s group is targeting all these problems, in a very specific way.”
Tinsley was immersed in the corrination research, building on some of the groundwork done by Jayme Workinger G’17, now a research scientist at Thermo Fisher, who earned a Ph.D. as a member of the Doyle group and was among the study’s co-authors. Tinsley handled the synthesis featured in the research paper and traveled to the University of Pennsylvania to work with the shrews used in the study. “We showed that we drastically improved the uptake of the corrinated compound versus the uncorrinated,” says Tinsley, who wants to pursue postdoctoral research and then design drugs for the pharmaceutical industry, with an interest in teaching one day. “I handled everything from the synthesis to working with the animals, to the selection of blood that’s used in the analysis, to doing the analysis itself.”
Tinsley believes there’s still a great deal to explore with the corrination compound’s application, including its potential use in small molecules, an aspect they’re investigating in the lab. “We can make this corrinated compound better, and that’s part of the process—you have this lead compound and now you want to optimize it,” he says. “Now that we have the corrination technology and it has been put out there, I think it provides something for other people in the field looking at medicinal chemistry to potentially improve what they’re designing as well.”
For Doyle, that’s all part of the process—from asking questions that haven’t been answered to ushering a drug through the gauntlet of trials and tests, hoping it succeeds and can make a difference in people’s lives. Doyle’s taking one step in that direction with his colleagues at Cantius Therapeutics. In a separate project, they’re partnering with a venture capital firm in California to develop a drug that specifically focuses on cachexia—a muscle-wasting and weakness syndrome associated with chronic diseases like cystic fibrosis—and chemotherapeutic-induced nausea and vomiting. “You want to stave off the disease before it progresses, but treating it can be very difficult,” Doyle says. “We have to make sure there are no red flags in terms of human toxicity. Then it could progress along—and from funding to use in humans, the goal would be 18 months.”