Using magnets to find new drugs: Inside UAB's high-field nuclear magnetic resonance facility

Most high-end lab equipment is inaccessible to the public eye, but one of UAB's most powerful drug-discovery tools is clearly visible from the Campus Green. The Central Alabama High Field Nuclear Magnetic Resonance Facility occupies a gleaming ground-floor space in the Chemistry Building. Its massive magnets give researchers invaluable insight into disease-causing proteins — and the data they need to find new ways to stop them.

The cover story of the latest issue of UAB Magazine features the Alabama Drug Discovery Alliance, a partnership between UAB and Southern Research Institute that aims to accelerate high-potential discoveries from the lab to patient-ready treatments. One key tool in that process is the Central Alabama High Field Nuclear Magnetic Resonance Facility, which opened in 2013. The Mix takes a closer look in this new feature.

Spin This Way

Each of the facility's NMR machines specializes in a different type of job, but the basic functioning is the same, explains NMR director N. Rama Krishna, Ph.D., UAB professor in the Department of Biochemistry and Molecular Genetics. The machines generate strong magnetic fields that polarize the tiny magnets in the nuclei of hydrogen atoms. “Then, using radiofrequency pulses, you can count all of the individual hydrogen atoms in a sample, which tells you what amino acids are present and how they are arranged in space,” Krishna says. And that’s precisely the information you need to create a detailed picture of a protein’s structure.

Mapping a protein's structure is crucial to understanding its function — and to finding ways to alter that function to treat disease. For instance, locating suitable "binding pockets" on a protein linked to brain cancer tells medicinal chemists how to design a drug to block (or enhance) that protein. "That's why NMR is one of the most versatile tools for drug-discovery research," Krishna says.

The bigger your magnet, the better images you can get. The centerpiece of the NMR facility is an 850 MHz Bruker BioSpin model, one of the largest in the South, which allows scientists to analyze structural data on even the largest proteins.

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Building a Better Drug

The 850 MHz machine can also accelerate the drug-discovery process "by allowing researchers to rapidly test new compounds they've developed in the lab," Krishna adds. Using a technique called saturation transfer difference NMR (STD-NMR), Krishna and his team can register the minute changes in signals from hydrogen atoms that occur when a compound binds to a protein. It would be nearly impossible to capture this interaction directly, he points out, because "it may last only a few microseconds." With STD-NMR, researchers can screen a number of potential drugs at once, then focus on the ones that show signs of binding to the target protein.

UAB's Rama Krishna and scientists from Southern Research
Institute have collaborated in developing a novel high-field
NMR-based protocol for determining the binding of
allosteric ligands to target proteins. They used the kinesin-5
protein Eg5 (a cancer target) and its inhibitor monastrol
 as an example (see above) for this protocol.

Using other techniques, researchers can analyze the disease-causing interaction between two proteins, and then find the right location to dock an inhibitor that would prevent the proteins from coming together. Or they could do the opposite, in an approach dubbed “fragment-based discovery” — using NMR data to identify two compounds that bind close together on a protein and “cross link” them to significantly improve their binding.

Krishna uses these techniques in his own National Cancer Institute-funded research to find new treatments for pancreatic cancer. Other UAB investigators are using the NMR facility to further their drug-discovery efforts in Parkinson's disease, brain tumors, breast cancer, heart disease, HIV and more. And as word of these capabilities has spread, researchers at institutions across the South have begun sending in samples to the NMR facility for evaluation.

Early Warning Signs

NMR is useful for many applications beyond drug discovery, Krishna adds. The facility's 600 MHz machine specializes in a hot area of medicine known as metabolomics, which studies the way the body processes everything from food to medicines.

"If you are taking a drug that is toxic to the liver, the body will generate some small molecules — known as metabolites — associated with liver damage,” Krishna explains. "We can detect these molecules in the NMR spectra of biofluids such as urine and blood plasma and say, 'Aha, after this patient started taking the drug, we can see an increase in these signals, so something is going wrong." That can warn researchers of side effects from new drug treatments "long before there is any major problem," Krishna says.

"The range of applications in this facility is amazing," adds Krishna. "It is a unique platform for everything from basic science to translational research.”

Unique gene machine opens new pathways to personalized medicine

In the UAB Nanostring Laboratory, researchers such as Eddy Yang are taking advantage of the nCounter Analysis System's novel digital profiling technology to examine specific signaling pathways in cancer and other diseases. That work could lead to new insights to improve diagnosis and treatment decisions.

Cancer is a devious enemy. In lab tests, researchers have identified plenty of exciting genetic targets — weak links that should allow them to destroy tumors by halting production of a crucial enzyme, for example, or blocking a signal the cell needs to keep growing. All too often, however, these promising findings fizzle out in further testing.

That is because cancer cells can take advantage of multiple, redundant signaling pathways to avoid areas that come under attack. “Tumor cells will just figure out a way to bypass them,” said Eddy Yang, M.D., Ph.D., an associate scientist in the UAB Comprehensive Cancer Center and associate professor in the UAB Department of Radiation Oncology.

Mapping out the complex pathways involved in cancer and other diseases is a crucial step in finding better treatments — and identifying the best treatments for individual patients. If you know all the routes a tumor can use to evade attack, you can find therapies — or combinations of therapies — to block them all. Indeed, tracing cancer-related signaling pathways, and finding ways to use these insights to improve diagnosis and treatment decisions, is a major focus of research at the Cancer Center, Yang says. But spotting these pathways amid the information overload of a genomewide screening test can be extremely complex and time-consuming.

nCounter: In Focus 48-800 genes can be studied simultaneously Applications include gene expression analysis, microRNA and lncRNA analysis, copy number variation analysis, ChIP-String analysis and leukemia fusion gene analysis The system's novel digital technology is based on direct multiplexed quantification of nucleic acids; it provides highly reproducible data over 5 logs of dynamic range Preconstructed panels include: PanCancer Pathways Panel, Human Kinase Panel, Human Immunology Panel, microRNA Panels, Cancer Copy Number Variation Assay See the UAB Nanostring Laboratory site for more information and a schedule of fees and services

Targeting Crucial Pathways

Now, UAB researchers and clinicians have a new tool to investigate signaling pathways — and to translate their discoveries into clinic-ready diagnostic tests. The unique nCounter Analysis System, produced by Nanostring Laboratories, “is a platform to measure the expression of genes in a targeted manner,” Yang said. “Instead of looking at the whole genome, you can investigate anywhere from 48-800 genes at a time.” Researchers can zero in on certain pathways that they are studying, Yang explains, or they can use preset panels of previously identified cancer networks.

Yang directs the new UAB Nanostring Laboratory, which is open to investigators across campus. He is using the nCounter to pursue his own research in experimental treatments for breast, prostate, and head and neck cancers. “I’m very interested in understanding the pathways that make a tumor tick,” Yang said. “I want to know which ones make it resistant to therapy and which ones could actually make it more sensitive to treatment.” With the nCounter, Yang said, “we can look from a 10,000-foot perspective rather than from sea level. It’s an exciting technology.”

Another advantage of the nCounter is that, unlike other technologies, which may require whole molecules of high-quality RNA or amplification for analysis, the nCounter can gather information from small pieces of RNA. That means researchers can use it to look at pathways in tissue that is up to several decades old, retroactively verifying new patterns they have found instead of having to collect new samples for analysis. The nCounter can also perform a range of other tests, Yang says, including analysis of microRNAs, gene fusions and gene amplifications.

From Concept to Clinic

The nCounter is more than a research tool. It can run new diagnostic tests such as the ProSigna Assay, which gives clinicians an estimate of a patient’s likelihood of tumor recurrence based on which pathways are active in that patient. It is an excellent example of personalized medicine in action, Yang says. He envisions UAB researchers using the nCounter to develop novel tests to inform treatment decisions in cancer and other diseases. UAB is one of the first institutions nationwide with the ability to do both laboratory and clinical testing using the nCounter.

“We hope to use the pattern of the pathway of genes to help guide therapy,” Yang said. “That’s the personalized medicine approach.”

Yang and collaborator Andres Forero, M.D., senior scientist at the UAB Cancer Center, will use the nCounter as part of a clinical trial testing a new treatment approach against triple-negative breast cancer. The trial, which recently began enrolling patients, is testing two different drugs — a PARP inhibitor and an EGFR inhibitor — to block two different pathways used by these tumors.

“By blocking PARP, you block the ability of the tumors to repair DNA damage,” which should eventually result in cell death, Yang explains. But the tumors can take advantage of an alternate backup pathway to repair that damage, meaning PARP inhibitors alone are often ineffective. “By blocking EGFR, we will block that backup pathway,” Yang said. [To learn more about this study, call (205) 934-0309; visit the Clinical Trials section of the Comprehensive Cancer Center's website to see all current studies.]

Using the nCounter, the researchers will compare the pathways altered in patients who respond to the therapy with those in patients who aren’t helped by the combination. In the future, that could let them identify the most appropriate patients for this treatment with a simple test. “We hope to use the pattern of the pathway of genes to help guide therapy,” Yang said. “That’s the personalized medicine approach.”

Grad student receives national award for new insight on alcohol and liver damage

Uduak Udoh

By connecting the dots between chronic drinking, molecular clocks, and energy storage patterns, UAB doctoral student Uduak Udoh has identified a potential new approach to target alcoholic liver disease. The work has also earned her a top honor from the Research Society on Alcoholism (RSA) and the National Institute on Alcohol Abuse and Alcoholism (NIAAA), and kudos from former NIAAA director Enoch Gordis, M.D.

Udoh, a fifth-year doctoral student in Pathobiology and Molecular Medicine, received the Enoch Gordis Research Recognition Award during the RSA's annual scientific meeting this summer. At the meeting, she presented results from her dissertation project, "Hepatic Glycogen Metabolism Is Impaired by Alcohol Consumption: Possible Role of the Liver Molecular Clock."

Liver cells, like almost all human cells, have a built-in circadian clock—a set of genes that control metabolism and other biological processes in a daily cycle. Udoh's research shows that chronic alcohol consumption disrupts the liver's normal pattern of creating glycogen, a storage form of glucose.

"Glycogen in the liver is an important fuel reserve that the body uses in between fasting and eating," explains Udoh, who is a member of the lab of Shannon Bailey, Ph.D., in the Division of Molecular and Cellular Pathology. Previous research has shown that the liver clock controls glycogen synthesis in a regular rhythm throughout the day. Emerging studies show that alcohol can disrupt the liver clock's timing, just as it disturbs the main circadian clock in the brain to disturb sleep and other behaviors.

Udoh's work connects these two observations. In mouse models, "we normally see a nice diurnal rhythm to glycogen content in the liver, which makes sense because metabolic needs vary throughout the day," Udoh says. But chronic alcohol consumption brings a significant change in that pattern, and a corresponding decrease in glycogen levels, Udoh found. She also demonstrated that alcohol disrupts signaling genes and proteins regulated by the liver clock that control glycogen metabolism.

Without sufficient glycogen, the liver may lack the energy to repair alcohol-generated damage, contributing to alcoholic liver disease. Ultimately, Udoh's research "highlights the molecular clock as a novel therapeutic target for alcoholic liver disease," says Bailey. (Learn more about Bailey’s own research into chronic alcohol consumption and the liver clock in this Mix podcast.)

Udoh's work was one of only six selected for presentation at the Research Society on Alcoholism meeting from hundreds of applications. Judges selected her for the Gordis award, which recognizes outstanding research among graduate students and postdoctoral fellows, based on her oral presentation and research poster session. One of the highlights of the event was discussing her work with Dr. Gordis himself, Udoh says. "It's a great honor."

Using 3D printers and movie modeling techniques, UAB researchers enhance workplace safety devices

Claudiu Lungu and a team from UAB's Department of Environmental Health Sciences have devised a high-tech, low-cost method for designing and fabricating new respirator prototypes to improve workplace safety.

If you work on an auto painting crew, stir vats of artificial butter at a popcorn factory or handle asbestos at a shipyard, you are one of the 5 million American workers legally required to wear respiratory protective equipment on the job.

But legal requirements and actual practice don't always match up. And even when workers wear their respirators, they may not be doing much good.

Studies show that hundreds of thousands of workers—from 15 to 20 percent, according to recent research—may be wearing ill-fitting respirators, not designed for a workforce that has rapidly changed over the past decades.

But a UAB research team has devised a high-tech, low-cost method for designing and fabricating new respirator prototypes to better match the variety of facial shapes in today's workplace. In addition to protecting industrial workers, the technology could aid members of the military as well. The findings are published online in the Journal of Occupational and Environmental Hygiene.

Superfoods and breast cancer: Study takes a closer look at broccoli and green tea

Could a combination of broccoli sprouts and green tea offer protection against breast cancer — and transform hard-to-treat breast tumors into a type that responds to medication?

A series of studies in the lab of UAB biologist Trygve Tollefsbol, Ph.D., D.O., have generated encouraging findings. Tollefsbol, who is also a senior scientist in the UAB Comprehensive Cancer Center, has shown that mice given sprouts in their chow and green tea polyphenols in their water are protected against tumor development. Intriguingly, he has also shown in animal studies that the combination can change estrogen receptor-negative (ER-) tumors, which have few treatment options, into estrogen receptor-positive (ER+) tumors, which can be treated with the anti-estrogen drug tamoxifen.

Now, Tollefsbol has received a $1.5-million, five-year grant from the National Institutes of Health to pinpoint the molecular mechanisms behind these effects. "We already have a lot of preliminary data showing that this combination works," Tollefsbol says. "The grant will allow us to extend that research and explore the effects genome-wide."

The immortality enzyme? Telomerase fights aging, fuels cancer

In a lab in the heart of Campbell Hall, UAB biologist Trygve Tollefsbol, Ph.D., D.O., stores the secret to immortality—but you may not want it.

Trygve Tollefsbol is a renowned expert on telomerase, an enzyme that
plays crucial roles in determining our lifespans and fueling cancer growth.

Telomerase, the enzyme in question, is a quirky character. Even though it is dormant most of the time, it appears to play a key role in all three of Tollefsbol’s main research interests: aging, cancer, and epigenetics.

Telomerase’s job is to lengthen telomeres, little caps at the end of our chromosomes that keep the chromosomes from becoming unstable during cell division. (They’re kind of like the plastic cylinders on the ends of shoelaces, Tollefsbol says.) But a little bit gets shaved off with each cycle of division. Eventually, there is very little protective telomere left, and cells age and stop dividing.

Truth and consequences: Building a game to fight the rural HIV epidemic

UAB researcher Comfort Enah is developing a video game to help high-risk teens and pre-teens
learn vital lessons about HIV prevention. An early graphic concept is shown above.

Comfort Enah, Ph.D., a researcher in the UAB School of Nursing, can't build a time machine to help teens avoid making bad decisions in the future. So she's creating the next best thing: a video game.

Working with a team from the School of Engineering, Enah is crafting a simulation of the challenges of modern teen life—including social media shaming, drug and alcohol use, dating boundaries, and the wildfire spread of misinformation on the Internet. The goal is to slow the HIV epidemic among adolescents in the rural South. Enah's dream, if the game proves effective, is to take it to the even more hard-hit communities of sub-Saharan Africa, where she grew up.

Maturity without Maturity

Over the past century, puberty has been arriving earlier and earlier, which means that “teens are spending longer and longer periods with bodies that are sexually mature and brains that aren't yet capable of anticipating the long-term consequences of their actions,” says Enah, an assistant professor in the Department of Nursing Community Health Outcomes. “They need to practice their responses to those risky situations, and games are a way to do that in private and as often as necessary.”