by Paige Brown
At LSU, the development of new medical drugs takes on many different shapes and forms, from the determination of new drug molecule structures that fit into human disease targets like a key in a lock, to high-throughput screening of natural plant product libraries for unknown drug properties. At the base of drug development is laboratory discovery, advanced measurements on state-of-the-art equipment and, most importantly, teamwork.
Structure is Everything
It all started in 1953, when James D. Watson, Francis Crick and Rosalind Franklin deciphered the double-helical structure of our hereditary material from a ring-like pattern on a picture of DNA taken with X-ray diffraction equipment like that found in LSU’s Center for Advanced Microstructures & Devices, or CAMD. The discovery of the structure of a complex three-dimensional structure such as DNA was a breakthrough for modern medicine, and opened up a whole new field of structural biology.
Marcia Newcomer, professor in the Division of Biochemistry and Molecular Biology within the Department of Biological Sciences at LSU, is now putting X-ray crystallography and other structural biology techniques to work for the development of new drugs for inflammatory diseases. Newcomer is using synchrotron radiation resources found at CAMD to decipher the shapes of molecules important for new drug development.
Using information about the shape of a drug target molecule can help researchers like Newcomer fashion drug compounds that stick to these drug targets and stop their disease-causing activities in the human body.
“One of the ideas behind drug design is that if you know what the molecules look like that you want to stop, you can make something that is specifically designed to jam them up, to stop them from working,” Newcomer said. “This is an important approach to drug design. Basically, you get a three-dimensional structure of a molecule and then you make something small, like a silver bullet, to take this molecule out. But you have to find a molecular structure that is a good drug target.”
Newcomer works on an enzyme called 5-Lipoxygenase, or 5-LOX, which initiates the synthesis of compounds called leukotrienes in the human body. Leukotrienes promote inflammation and inflammatory responses, for example, swelling and contractions of airway muscles in asthma patients.
“That is part of the normal inflammatory response to help you get rid of microbes and other dangerous foreign bodies,” Newcomer said. “But you also need to shut it down in some cases before it causes problems.”
There are several problems associated with a heightened inflammatory response and leukotriene production, including asthma. Newcomer’s enzyme of interest, 5-LOX, is crucial in the pathway that produces leukotrienes in the human body, and thus is a prime target for drug inhibitors. Unfortunately, 5-LOX is also an unstable enzyme and very difficult to work with. The pharmaceutical company MERCK had tried for many years to determine this enzyme’s structure with the goal of fashioning new drugs to hinder inflammation based on its shape, but finally gave up the pursuit.
Then, in 2011, Newcomer and colleagues were the first to successfully decipher the crystal structure of the enzyme in research that was published in the prestigious Science journal. Using creative strategies to stabilize the fickle 5-LOX, Newcomer’s group was able to create high-quality crystals of the active enzyme. Using these crystals, the group could get clear pictures of the enzyme’s three-dimensional shape using X-ray crystallography.
According to scientists who have since written reviews of Newcomer and colleagues’ groundbreaking work, the new structural information opens doors for discovery and development of new therapeutics for inflammatory diseases and ailments such as asthma, cardiovascular disease and even cancer.
One of the first drugs developed to treat the AIDS virus was developed in this way.
“Scientists got a structure of one of the molecules that is necessary for the AIDS virus to replicate, and then they made something that would bind this molecule and stop it from accomplishing what it’s supposed to do in the lifecycle of the virus,” Newcomer said.
Molecular structure is of vast importance when it comes to how drugs work against disease targets in the human body. For example, the antibiotic penicillin works because its shape allows it to bind to the bacterial enzyme that helps a bacterium make its own cell wall.
“If the bacterium can’t make its own cell wall anymore, then it can’t survive, thus the antibacterial properties of penicillin,” Newcomer said.
Newcomer and her colleagues at LSU work on the structural biology end of drug development, working to determine the structures of target disease molecules like 5-LOX in order to provide this information to other researchers who can make drugs that bind to and inhibit the action of these molecules.
“We make it possible for other people, including pharmaceutical chemists, to use this structure to develop drug inhibitors,” Newcomer said.
However, determining the three-dimensional structure of a molecule like 5-LOX is not as simple as looking through a microscope. The molecules Newcomer and her colleagues study are very small – so small that the light rays that normally illuminate the objects we can see in our macro world can’t be used to visualize them. The structure of molecules must be determined using analysis equipment that uses X-rays to determine three-dimensional shape.
Having LSU’s CAMD facilities nearby has been a major asset to Newcomer’s research. The synchrotron particle accelerator at CAMD can produce many different types of X-rays required for three-dimensional crystallography. While researchers can produce the radiation needed for molecular structure analysis in home laboratory sources, the rays generated from these sources are not very bright, and they only consist of one color, or wavelength of light. A synchrotron produces white radiation, which is a combination of different wavelengths, or colors, of X-rays.
“If you think about light being a spectrum of colors, home lab X-ray sources give you only one color,” Newcomer said. “But there are experiments for which you need more than one color. For these, you need to go to a synchrotron source. You can’t get three-dimensional structure information without it.”
“CAMD is one of only six facilities in the United States that you can do this kind of work at,” Newcomer said. “While our group still gets some data from the brightest synchrotron near Chicago [Advanced Photon Source at Argonne National Laboratory], we still use CAMD for all of our preliminary work. This makes our work go so much faster, because if we had to organize around when we can get time in Chicago, it would take years to do what we can do now in much shorter time.”
Newcomer has always worked on molecular structures. One of the reasons she went into the structural biology field was the promise that someday it could be helpful in drug design. Now, with the crystal structure of 5-LOX determined by Newcomer’s group, structural biologists and medicinal chemists can team up to design new enzyme inhibitor therapeutics.
Just as 5-LOX catalyzes the synthesis of inflammatory molecules in the human body, Newcomer’s research, along with her use of unique resources available at LSU, has served as a catalyst for new efforts to produce drugs for severe inflammatory diseases.
A Team Approach
Today, Grover Waldrop, adjunct professor in the LSU Department of Chemistry and professor in the Department of Biological Sciences, has assembled a team of LSU researchers spanning the fields of chemistry, microbiology and computational studies to fight a particularly nasty problem: antibiotic-resistant bacteria.
In 1995, Waldrop began work on acetyl CoA carboxylase, or ACCase, an enzyme involved in metabolism of fatty acids in animals, plants and bacteria. ACCase is a target for drugs to help fight obesity and for drugs that kill bacteria, or antibiotics, where bacteria will not grow if deprived of this enzyme. In 2004, Waldrop’s groundbreaking work with the bacterial form of ACCase, which is involved in the making of bacterial cell membranes, akin to the “skin” protecting bacteria from their outside environment, earned him an antibiotic development partnership with Pfizer, the world’s largest research-based pharmaceutical company.
“I learned a lot about pharmaceutical development, of which I knew nothing,” Waldrop said. “We developed some molecules that targeted my enzyme and had antibacterial activity.”
Unfortunately, in 2008, Pfizer discontinued their entire antibiotic drug development division.
“The reason they dropped all of their antibiotic projects is because antibiotics are not profitable,” Waldrop said. “Yet there is a looming public health care crisis because of the dramatic rise in antibiotic resistant bacteria. This is one example where a market-driven approach does not reach the best solution.”
Meanwhile, Waldrop’s colleagues at Pfizer encouraged him to continue his research path.
“They felt that there was a clear medical need for this type of work,” Waldrop said. “So they sent me all of their chemicals and materials. In a single day, we received seven large boxes. It was like Christmas!”
The end of Waldrop’s collaboration with Pfizer catalyzed a new direction in his work, one that harnesses a more multi-disciplinary and team-based academic approach. While industry is limited by economic responsibilities, Waldrop believes there is a niche for an academic approach to antibiotic development.
“One laboratory can’t develop a drug,” Waldrop said. “Everybody needs to work together to achieve a single goal. Where you involve multiple people, you have more chance of success.”
Waldrop and his collaborators Carol Taylor, associate professor in the LSU Department of Chemistry; Greg Pettis, associate professor in the LSU Department of Biological Sciences; and Michal Brylinksi , joint assistant professor in the LSU Department of Biological Sciences and the Center for Computation and Technology, or CCT, are now pursuing what Waldrop calls a two-pronged approach to antibiotic development. This approach involves searching for both synthetic and natural products that can target his enzyme and have antibiotic properties.
In collaboration with Taylor’s organic synthesis laboratory, Waldrop’s group is constructing a synthetic prototype for a new antibiotic. The collaborative project analyzes the properties of known ACCase inhibitors that have been transformed in an effort to improve their ability to bind to the bacterial enzyme (ACCase).
Waldrop also collaborates with Brylinski, who works on the virtual side to discover new drug targets via computer modeling. Modern drug discovery is strongly supported by computational techniques, which can help identify new potential drug compounds. Brylinksi’s group can screen through large libraries of virtual compounds to determine promising inhibitors of Waldrop’s bacterial enzyme. The calculations, which require substantial computing power, will be carried out on a new LSU high-performance computer cluster provided by High Performance Computing at LSU, or HPC@LSU, a partnership between LSU’s Information Technology Services and CCT.
“The goal is to use computational modeling to limit the size of the screening library to those compounds that most likely exhibit the desired biological activity,” Brylinski said. “In this project, we will evaluate millions of compounds in silico [i.e. via computer simulation] prior to experimental screens, at a fraction of the cost.”
Armed with computational screening and organic synthesis resources, Waldrop’s lab is building up compounds that prevent bacteria from making their own cell walls. By determining the three-dimensional shape of ACCase and potential inhibitor targets via X-ray crystallography at LSU’s CAMD facilities, Waldrop and his colleagues can build, from the ground up, inhibitor drugs that fit lock-in-key inside the active site of the enzyme.
However, Waldrop isn’t stopping with synthetic products. He is also going down the unbeaten path of natural product screening for antibiotic development.
“I learned that Pfizer didn’t screen for natural products,” Waldrop said. “So now, our lab does natural product screening. Many botanical products may be active as antibacterials.”
In collaboration with the Botanical Research Center at Pennington Biomedical Research Center, Waldrop’s group recently screened his ACCase against a collection of natural products and found an unusual target: cranberries.
“Cranberry extract inhibited my enzyme,” Waldrop said. “Cranberries – who would have known?”
According to Waldrop, the LSU Herbarium, a facility that houses more than 180,000 plant specimens, could be a gold mine for antibiotic screening.
“There is a lot of potential there,” Waldrop said. “We are looking for molecules that can be used as drugs against bacteria. This is truly applied research.”
Finally, all new antibiotic prototypes developed by Waldrop’s team, whether synthetic or natural, can be examined for antibiotic activity against known bacterial pathogens in Pettis’ microbiology laboratory.
“We could never do this work without our organic chemistry resources in Taylor’s lab and Pettis’ measurements of microbial activity,” Waldrop said. “It’s not just my lab. My work benefits from the diverse resources available at LSU, including synchrotron radiation available at CAMD, thousands of specimens at the LSU herbarium and computational screening abilities.”
One of the project’s most invaluable resources, however, is teamwork.
“Had Pfizer not called me, and had I never collaborated with them, I would never have realized the importance of teamwork,” Waldrop said. “I learned that a team approach is the only way that you are going to succeed, with everyone working together on a single goal. I’m trying to do that here at LSU.”
Waldrop is trying to foster teamwork at LSU, encouraging his colleagues to increasingly take this approach.
“It’s the same concept as taking a sports team mentality,” Waldrop said. “A person who is not a team player can spoil a whole game. Everybody is playing their role here, and we are slowly making progress.”
According to Waldrop, drug development research at LSU is like training for the big game: You have many more failures than you do successes, but in the end, teamwork is everything.