Basic Scientists in Search
of Clinical Answers

Victor C.Yang
Albert B. Prescott Professor of Pharmaceutical Sciences
Department of Pharmaceutical Sciences
E–mail: vcyang@umich.edu

Victor Yang has two favorite quotes he likes to cite to the graduate students, postdocs, and visiting researchers who work in his Michigan College of Pharmacy laboratories. The first: “Imagination is more important than knowledge,” attributed to physicist Albert Einstein. The second: “Do not join the flow, lead the flow.” That is Yang’s own creation, encapsulating a research philosophy honed over a 21–year career at the College.

“If you join the flow, you will become obsolete,” Yang explains. “If you lead the flow, you can set the direction. Leading is always more exciting, more intellectually challenging and — as I tell my graduate students — offers greater long–term job security.”

There is probably no faculty member at the College who has more diverse, applications– oriented research projects in progress, or has done a consistently better job securing coveted National Institutes of Health (NIH) funding than Victor Yang. (For a summary of the research projects now under investigation by Yang’s Michigan Pharmacy labs, see the accompanying sidebar, page 6.) Supported non–stop by NIH grants since he joined the College in 1986, Yang’s NIH funding now totals more than $15 million with approximately $5 million in grants spread over the next five years. These grants span areas of drug therapy ranging from bioreactor–based detoxification to innovative drug delivery systems including transdermal, targeted, and prodrug delivery approaches, and synchronized magnetic resonance (MR) imaging and drug chemotherapy to treat various types of cancers. What’s more remarkable is that only one of the seven NIH–funded research projects now being pursued by Yang’s lab predates the six–month sabbatical leave he took in 2001. That sabbatical, he says, was a pivotal point in his research career.

Professor Victor C.Yang (center) and his U–M College of Pharmacy research group.

“When you work in a large, complex organization like U–M, you become so occupied getting done what needs to get done that you lose touch with important scientific discoveries taking place all around you,” Yang explains.

“That runs counter to creative thinking because you have no new knowledge beyond your current research. I had individual projects that had been funded continuously by NIH for 14 years. We were productive and being rewarded for it. I could have continued refining what we had already done and continued getting funding, but I wanted new challenges, new ideas to work on, to refine, to develop. But first, I needed new knowledge to give form to these challenges.”

A man on a mission, Yang spent the first half of his six–month sabbatical at MIT — where he had, from 1983 to 1985, been a postdoctoral research associate in the Department of Chemical Engineering laboratory of biotechnology entrepreneur and visionary Robert Langer — and the final three months in Japan. Yang practically lived in science and engineering libraries, reading six technology papers a day, 30 days a month, for six months. By the time he returned, he had “tons of exciting new ideas, and much better understanding of new technologies,” Yang says.

One of these new ideas recently received a $1.28 million, fiveyear NIH grant. With this grant, Yang and his research team will use natural biomolecules to deliver medication in two parts, making it more specifically lethal to select cancer cells while leaving healthy cells and molecules unharmed.

“Two of the biggest obstacles researchers face in developing new treatments for cancer are a lack of selectivity in the cells cancer drugs intend to kill, and difficulty getting drugs through the cell membrane of the tumor cells,” Yang explains. “We can add a targeting component to the drug and direct it to the liver to solve this selectivity problem, for example, but that doesn’t solve the problem of getting the drug into the diseased cells while leaving healthy cells alone.”

Yang’s idea is to give the patient an inactive drug that zeros in on the target cells. He then injects a separate compound that activates the drug when it reaches its target, not before. This drug delivery method can also produce precise magnetic resonance images (MRI) of tumor tissues.

He is using a combination of cell–penetrating amino acids called the TAT peptide as a carrier molecule. This positively charged molecule normally carries a protein across the cell’s membrane, which has a negative charge. After administering an activating compound, the TAT peptide gets the attached drug molecules into the interior of the targeted cells — first the inactivated TAT drug complexes, and then the activating compound that turns the TAT–drug system on.

Figure 1. An image of the magnetic nanoparticles used by Victor Yang’s research group as a drug carrier for brain tumor imaging and therapy. Image courtesy Victor Yang.

The strategy works in experiments, though Yang said scientists are still unclear on how the peptide can get into the cell. Since the TAT peptide is based upon HIV, he speculates that it may use the same mechanism as a virus does to invade a cell.

Yang hopes to use a similar targeted approach on brain cancer, one of the most difficult cancers to treat with chemotherapeutic agents.

“The blood–brain barrier is a nearly impervious membrane, which greatly limits our capacity to attack brain tumors via the bloodstream with conventional medications,” Yang states. “Traditional chemotherapeutic agents tend to be molecules too large to cross the barrier. Our line of attack is to target these cells with the TAT peptide carrier in order to both improve tumor imaging while simultaneously delivering a therapeutic dose of medicine into the brain tumors.

“Solving the blood–brain membrane barrier problem is paramount. If I can solve this problem, I believe I can solve any other kind of tumor problem.”

His aim is to arrive at a treatment that combines both the targeting and the drug therapy in one delivery system.

In recognition of his wide–ranging academic and research achievements, Yang was named a Cheung Kong Scholar by the Chinese Ministry of Education (CMoE) in 2005. The main objective of the Cheung Kong Scholars Program is to advance China’s educational and scientific competitiveness through training the country’s best young scholars. Consequent to receiving this prestigious award, Yang received an adjunct faculty appointment at Tianjin University’s School of Chemical Engineering. CMoE subsequently provided funds to the School of Chemical Engineering in order for Yang to establish a laboratory there. Yang will discuss the scientific and strategic implications of his Cheung Kong Scholar appointment in the Spring 2007 issue of Interactions.

Professor Ronald W.Woodard

Ronald W.Woodard
Chair and Professor
Department of Medicinal Chemistry
E–mail: rww@umich.edu

The decades–long rise in antibiotic resistance has taken some of the luster off one of the 20th century’s true families of wonder drugs. Penicillin, once effective against most staph strains, is now nearly useless against this pernicious microbe. Methicillin, amoxicillin, and related drugs, developed to combat penicillin–resistant staph, also are increasingly ineffective against many staph infections. Staphylococcus aureus, a major source of hospital–acquired infections, has shown signs that it, too, may be developing resistance even to vancomycin.

“Every time science invents a new way to trick them, bacteria find a detour, usually within a few bacterial generations,” notes Ronald Woodard, chair and professor of the College’s Department of Medicinal Chemistry. “On top of that, drug–resistant genes from one microbe are often exchanged with different microbes. So we’re in this running battle with lethal microbes and losing ground because our enemy can change shape and direction so quickly. We can’t invent new antibiotics fast enough to keep up with the innate ingenuity of the bugs.”

But what if science could find more efficient ways to use existing antibiotics, or develop new attack strategies that would lead to new, more effective antibiotics?

Woodard’s research team may have done just that by genetically modifying a strain of Gram–negative bacterium Escherichia coli (E. coli ) so that it lacks its normal outer protective layer of hair–like lipopolysaccharides (LPS), complex sugar and carbohydrate structures that help them defend against antibiotic attack. Without this layer, E. coli and other Gram–negative bacteria are more vulnerable to antibiotic assault.

Some of the better–known Gramnegative bacteria are Salmonella typhi (typhoid fever and foodborne illness), Neisseria gonorrhea, Vibrio cholerae, and Neisseria meningitidis (meningitis), along with Yersinia pestis, the bacterium implicated in the Black Plague.

Figure 2. Panels A and B are highly magnified electron microscope images of a normal E. coli cell. B is a close–up of panel A with labels of the various important cell layers and regions. Panels C and D are highly magnified electron microscope images showing the new E. coli cell created in Woodard’s lab, with D being a close–up of panel C. The outer membrane vesicle (OMV) formations in panel C indicate that the innards of the new cell have begun to protrude, which should make the cell easier to kill, if it can even survive. Panel D also shows some major differences in the various important layers and regions of the knockout E. coli. Image courtesy Ronald Woodard.

After stripping away E. coli’s LPS through genetic manipulation, Woodard’s research team found that E. coli was killed with just a fraction of the antibiotic dose typically needed. The E. coli knockout was 512 times more susceptible to Rifampin, 256 times more vulnerable to Novobiocin, and eight times more susceptible to Bacitracin, suggesting doses could be dramatically cut and still be effective.

Antibiotics typically only effective against Gram–positive bacteria also may be effective against Gram–negative bacteria stripped of their LPS defenses, Woodard adds. Gram–positive bacteria, generally considered easier to kill with antibiotics, include anthrax and other strains that cause upper respiratory infections and sepsis.

Interestingly, the discovery by Woodard’s research group came about indirectly.

“We set out to genetically modify the cells to eliminate the key sugar, which anchors LPS to the cell’s exterior,” Woodard explains. “We did that, but, to our surprise, the bug didn’t die.”

His researcher team found that a “backup” gene from a different pathway also could form the anchor, so they knocked out that gene, as well. Initially the cell with both genomic knockouts did not survive without special nutritional supplements. Later, they were surprised to see that with different growth conditions, the cells survived.

“Science dogma for the last 100 years was that Gram–negative cells could not survive without the external LPS structures,” Woodard states. “We discovered, much to our amazement, that this is not true.

“We also found that the genetically modified E. coli would die in the presence of normal levels of bile salts it would be exposed to in the human gut. (Naturally occurring E. coli can typically withstand the bile salts found in the human digestive tract.) Thus, by knocking out the E. coli genes that instruct the cell how to manufacture LPS structures, we weakened the bacteria’s ability to protect itself from the human body’s basic defenses.”

Woodard says his research team originally was disappointed when the Gram–negative E. coli wasn’t dying because they figured it had become resistant — that it had learned how to remake the LPS without the original blueprint, using a different method. In actuality, the bacteria discovered it could live without the LPS strings, at least in the controlled conditions of a laboratory.

Knowing that his lab’s discovery was completely contrary to conventional wisdom on bacteria — that is, that a Gram–negative bacteria could survive without LPS — Woodard solicited second opinions from the Borstel Research Center in Germany, known for their expertise in Gram–negative bacteria. Scientists there were initially skeptical, he notes, but eventually, Uwe Mamat and later Buko Lindner from Borstel signed on to the project and became co–authors of a paper published in the Feb. 17, 2006, print issue of ACS Chemical Biology, the new flagship monthly publication of the American Chemical Society.

Timothy C. Meredith, PhD’06, who co–authored the paper, was Woodard’s doctoral student when the research discovery was made. Meredith is now a postdoctoral researcher working with Suzanne Walker at Harvard Medical School’s Department of Microbiology and Molecular Genetics. Parag Aggarwal, PhD’06, also contributed experimental data for the paper.

“Tim was the driving force behind the initial discovery,” Woodard says. “The knockout was his work. He was the one who recognized the anomalies in the modified E. coli strain, and was the first to grasp the scientific implications.”

So, where does Woodard go, now, with the proof–of–concept discovered in his lab?

“Our goal was to identify strategic targets that would enable us to genetically cripple E. coli’s capacity to defend itself,” Woodard states. “We’ve proven that we can inhibit the enzyme that controls LPS development with E. coli, and by extension, any other Gram–negative bacteria. To that extent, it’s ‘mission accomplished.’ As an academic researcher with a comparatively small research group and budget — and all academic research groups and budgets are small by comparison with what you’d find in industry — we lack the resources to pursue this investigation further. That’s the job of industry.

“By contributing to an improved understanding of bacterial resistance machinery, our hope is that industry can build upon our findings to create new and better antibiotics. But even if they jumped on the bandwagon today, they would still be many years away from rolling out a product for human testing and clinical use,” Woodard adds.

“Nature has equipped bacteria with the means to circumvent even the latest and greatest approaches. As a human being, there’s nothing I would like more than to see science gain the upper hand on microbes. Hopefully, we’ve contributed to that.”

In addition to the information gained in discovering potential new targets for antibiotic/antimicrobial agents, Woodard also sees other humanitarian applications flowing from use of the new E. coli cell line created in his lab. (See Figure 2, panels C and D.) “Our new E. coli cell line is being patented by the University because of its potential to produce less–expensive, lower–toxicity vaccines and human protein products, such as insulin and human growth factor,” Woodard notes. “In the short term, it’s one way we can maximize our discovery to benefit humanity.”