The Berkeley Science Review: Articles

Neglected No More
Tropical disease research at UC Berkeley (view PDF)
by Niranjana Nagarajan

You get bitten by a tsetse fly, fall ill, then fall asleep and never wake up.

Sleeping sickness is a scary disease, right out of a horror film. Or somewhere, somehow, you breathe in one bacterium, called Mycobacterium tuberculosis, and you're stuck with it for life. It lives inside your body, hiding and protecting itself until the right moment, when it starts to multiply, ravaging your immune system.

Sleeping sickness, or human African trypanosomiasis, affects close to 500,000 people a year in Africa, killing 66,000. In 2006, 14.4 million people worldwide carried tuberculosis; 1.5 million died from it. And still, these diseases are neglected, largely under-represented in the academic world of medical research. UC Berkeley stands out, despite having no medical school, with a host of research programs on campus working on finding treatments and vaccines for neglected diseases.

Targeting trypanosomes
UC Berkeley professor Matt Welch, in the Department of Molecular and Cell Biology, works on identifying new drug targets in trypanosomes, the parasites that cause sleeping sickness and Chagas' disease (American trypanosomiasis, a disease prevalent in Central and South America in which a similar parasite causes damage to the heart and digestive tract). Welch's lab studies the cytoskeleton, a framework of proteins that holds cells together and gives them their structure. A few years ago, Robert Douglas, then a consultant for Cytokinetics, a biotech company specializing in drugs that target the cytoskeleton, approached him about setting up a research program to identify possible drug targets in trypanosomes. "[Douglas] decided that he wanted to work in neglected diseases," Welch says, "and he couldn't do that in the context of the biotech industry," because "that's not something the biotech companies are interested in, because there's no money to be made." As a result, Welch and his lab entered the realm of neglected disease research.

The cytoskeleton maintains cell structure by forming a complex network of interconnected strings of proteins, called filaments and tubules. These filaments and tubules also serve as intracellular highways, along which many essential components move from one part of the cell to another. As a result, drugs that disrupt the trypanosome cytoskeleton could potentially make good therapeutics for sleeping sickness and Chagas' disease. Unfortunately, the major components of trypanosomes' cytoskeletons are extremely similar to our own, so such drugs would be toxic to us. Instead, a sub-family of cytoskeletal proteins, called kinesins, might make better drug targets since they vary a lot more between humans and trypanosomes. If the cytoskeleton forms cellular highways, then kinesins, or motor proteins, are the trucks that transport essential materials via this network.

At the same time that Douglas and Welch began collaborating, the trypanosome genome sequence was released. An examination of the sequence yielded a surprising result: trypanosomes have about 45 kinesins, an unusually large number (almost as many as humans have, unlike yeast, which has six). Together with UC Berkeley professor Zac Cande and then postdoctoral fellow Scott Dawson, they analyzed these 45 genes and were surprised again: while many of the trypanosome kinesins fit easily into previously defined families, some of them appeared entirely unique, and some that Welch and Douglas thought would be necessary for cell division (and therefore good drug targets) were missing.

"So that left us with the more difficult task of trying to identify what kinesins might be important for the physiology of trypanosomes and that would also represent drug targets," says Welch. The researchers have been going about the process methodically, blocking the production of each of the 45 kinesins one by one and determining which are essential for the trypanosomes' survival. So far, they have found four that affect trypanosome survival and are following up on one particularly promising target. When this kinesin is blocked, the trypanosome swells and eventually dies, apparently unable to divide. They cut the candidate kinesin into pieces, hypothesizing that it was the actual motor portion of this motor protein that was essential. Neatly enough, Cytokinetics, the company that Douglas consulted for, specializes in making small molecules that target this motor activity and is now screening their chemical libraries for compounds that inhibit this particular piece of the trypanosome kinesin.

Welch believes that this joint venture between his lab and Cytokinetics "speaks to the desire of biotech companies, even when it's not something directly in their product line, to participate in work like this, to try to identify drugs for these neglected diseases, even though they wouldn't be involved in marketing the drugs ultimately. The vision is [that] the company is a collaborator in this process and we've negotiated a situation where there are not a lot of intellectual property constraints on compounds that would come out of these particular studies."

Controlling tuberculosis
Trypanosomes are disease-causing organisms that live outside host cells, making them extracellular pathogens. Intracellular pathogens are disease-causing organisms that enter the cells of our bodies and live there, flying under the radar of the host immune system. "[Intracellular pathogens] are particularly hard to make vaccines against because they hide inside host cells," says Professor of Molecular and Cell Biology Tom Alber, who studies Mycobacterium tuberculosis, the bacterium that causes tuberculosis and an intracellular pathogen.

Humans have been suffering and dying from tuberculosis for centuries. One of the enduring mysteries of M. tuberculosis concerns its unique life cycle—it infects and lives untouched inside host cells called macrophages, which just happen to be important members of the host immune system. Macrophages are hungry cells, floating through the body and sampling their surroundings by swallowing pieces of whatever is around. Anything the macrophage swallows, including lurking M. tuberculosis, ends up in a special compartment called the phagosome. The cell generally fuses the phagosome with another cellular compartment called the lysosome. Lysosomes are centers of cellular destruction, with highly acidic interiors and a plethora of destructive enzymes. Once a phagosome fuses with a lysosome, the contents of the phagosome are chewed, corroded, and cut up into oblivion. M. tuberculosis neatly sidesteps all this destruction by blocking the fusion of its resident phagosome with lysosomes.

Lisa Prach, a fifth-year graduate student in the Alber lab, is interested in how exactly M. tuberculosis blocks phagosome-lysosome fusion. Her research was inspired by a study from a group at Cornell University that randomly deactivated genes in the bacterium and looked for those that were necessary for phagosome-lysosome fusion. One of the genes identified encodes an enzyme called diterpene cyclase, necessary to make a class of lipids called diterpenes, which seemed out of place in a bacterium. "This is a pretty crazy class of enzymes," Prach explains, "because they are really common in plants, and there's only one bacteria known to even encode this sort of thing. It's really unusual for TB to have something like this." M. tuberculosis makes this unique, unusual lipid and also carries out the really unusual function of blocking phagosome-lysosome fusion. Extend the thought to the observation that the enzyme that makes this unusual lipid is necessary for the unusual function of the bacterium, and you may have a big clue to the big mystery of how the bacterium actually blocks phagosome- lysosome fusion.

Prach then noticed that the different strains of M. tuberculosis used in the Alber lab, some of which are more virulent than the others, have different levels of diterpenes. She hypothesized that the amount of diterpene produced by the M. tuberculosis strains may affect virulence. So she is now in South Africa, with a travel fellowship from UC Berkeley's Center for Emerging and Neglected Diseases, in a lab that studies the epidemiology of tuberculosis. In South Africa, the Van Helden lab and collaborators have collected about 20,000 M. tuberculosis samples from patients in clinics outside Cape Town and in other parts of South Africa. They have detailed records of the important properties of the strains they have collected—what specific sub-type of bacteria they are, how they behave in mouse infection, whether the patients cleared the bacteria, and whether the bacteria were drug resistant. Prach wants to find out if she can "correlate the levels of different lipids, specifically diterpenes, in these various strains with disease progression and disease outcomes." Specifically, she wants to know if the diterpenes in M. tuberculosis are related to the virulence and drug-resistance of the bacteria.

Other work in the Alber lab deals with how M. tuberculosis signals to the host cell to leave it alone in its phagosome home. One of the best-known systems of signaling in mammals is the kinase phosphatase system. Enzymes called kinases (not to be confused with kinesins) add a specific chemical group, called a phosphate group, to proteins, which switches them on. Phosphatases, by removing phosphate groups, serve as off switches. Alber says that nothing was known about these systems in bacteria before his lab started their research. "What we're interested in is, what are the signals the bacteria sense, how does that signal from outside the cell switch on the kinase on the inside of the cell, and once the kinase is switched on, how does it change the physiology of the cell."

So what turns on the kinases? The Alber lab has showed that the kinases themselves, in M. tuberculosis and other bacteria, were switched on by pairing with each other. These kinases went on to turn on a number of other proteins in the bacterium, among them some called sigma factors, which decide what genes will be turned on and when. These proteins could be promising drug targets, given that they have functions that are so essential for bacterial life.

Another exciting potential drug target is a sugar export channel in the bacterium. Deleting this channel entirely is lethal for the bacterium, and a partially defective mutant is immediately recognized and cleared by an infected mouse, unlike intact bacteria. Alber points out that of all the proteins in M. tuberculosis, "this [channel] is among the 20 that the bacterium most requires the function of." The Alber lab has data that suggest a bacterial kinase may be controlling the activity of the channel. "We're trying to discover, how does the channel change when phosphorylated? What does the channel do? What does it export? And what on the outside controls this export?"

How promising is this protein as a drug target? Alber says, "We just made a presentation to the Global Alliance for TB drug development. We're hopeful that they'll support the development of a new drug and we'll see how far we can take it."

The Center for Emerging and Neglected Diseases
Given that there is such good, relevant research on neglected diseases on campus, it is surprising that UC Berkeley is not very well known for this kind of work. Part of the reason is that many of the labs on campus that work on aspects of neglected diseases are found in different departments and physical locations.

What the UC Berkeley campus needs is a way to enable researchers from different departments, but with shared interests in neglected diseases, to find each other and potentially work together. The Center for Emerging and Neglected Diseases (CEND), which officially opened in May of this year, aims to do exactly that. CEND is a coalition of campus researchers from 12 departments and colleges whose mission is to promote the development of better "health outcomes" for people that suffer from neglected diseases. The new approach is multidisciplinary. Alber, the director of the new center, explains that CEND's function is "to bring people together that work on different pathogens to look for common mechanisms of disease, common solutions to diseases. And to bring people together from different fields so we can come up with innovative solutions for the problems each person is facing within their research on a specific disease."

Temina Madon, the executive director, tells the story of CEND's origins. In 2005, Professor W. Geoffrey Owen, then Dean of Biological Sciences, set up a series of community meetings with anyone interested in neglected diseases. More people attended each successive gathering, until, Madon says, "eventually they reached a large enough community on campus that it was decided to start raising funds for a Center, to identify priorities, programmatic activities, and needs of the research community on campus."

Now, "CEND is bringing big discovery science into global health," says Madon. She says CEND has three primary goals: stimulating innovative basic research into potential drug targets; training, of both Berkeley students and students from developing countries; and translating basic research into drugs for neglected diseases.

Research, training, and translation
Drug discovery is a complex process, starting with basic research that identifies suitable drug targets, followed by validation of these potential targets, and culminating in the development of the medicine to treat a disease. UC Berkeley is a large campus, with many diverse areas of research, and as such is well qualified to carry out all the steps of the drug development process—biological research into the hows and whys of a disease, chemistry research into innovative ways of making drugs, and a pioneering approach to intellectual property, embodied in its humanitarian licensing policy, all on the same campus. CEND facilitates contact between researchers who have similar interests but may not have encountered each other otherwise. To this end, CEND has recently set up an infectious disease supergroup, where campus researchers can get together to discuss their research interests.

Another aspect of CEND's mission is training, both of Berkeley students and of students from developing countries. As Madon says, one of the aims of CEND is "to create biological scientists coming out of UC Berkeley who understand not just basic science but also [have] an understanding of intellectual property and how that affects access to the medicines, vaccines, diagnostics they may one day produce. We also want these Berkeley students to have had some experience working in developing countries so that they understand the unique challenges that you might face in developing a treatment or pursuing a research program that has impact in those settings."

CEND also has programs to promote collaboration between Berkeley researchers and students in other countries, with programs involving the Indian Institute of Technology (IIT) and the South African Center for Excellence in Epidemiology Modeling and Analysis (SACEMA). This summer, 11 students from the IIT spent some time at UC Berkeley in CEND and Energy Biosciences Institute labs researching neglected diseases and biofuels. SACEMA was originally founded by UC Berkeley School of Public Health Professor Wayne Getz independently of CEND, but it is now affiliated with CEND. SACEMA trains African mathematics students in epidemiology, and, as Madon says, "what's nice and unique about it is that it's not the extractive mining approach to research, where you collect data from the country but you take the data out of the country and model it in your lab in the US...you're actually empowering people locally to do the same kind of research that you do in collaboration with you."

Finally, an important function of CEND is actually getting potential drug candidates out into the market. While both Welch and Alber have established potential ways to transfer their research to a clinical setting, it is not as easy for many other academic researchers. Madon describes one of her roles as facilitating the interaction between the researchers who come up with the drug targets and the companies that would end up manufacturing them: "We've sort of relied on Pharma or Biotech to come to us and say ‘Oh, we liked that patent and we want to license it,' but now CEND, working with [UC Berkeley's intellectual property guru] Carol Mimura, is trying to create that glue between private sector, non-university, non-ivory tower players and campus people who are interested in moving their research into the world where it will have an impact. We are actually shopping our intellectual property around, instead of waiting for companies to come to us."

CEND also enables researchers to get feedback about the relevance of their research. The Drugs for Neglected Diseases Initiative (DNDI) is a non-profit product development organization that promotes not-for-profit partnerships between private industry and academic research that leads to drugs for neglected diseases. Feedback from industry in the early stages of research is useful, and CEND has organized meetings between DNDI and researchers who may have potential drug targets where DNDI members had suggestions to make UC Berkeley research more relevant to treatment of neglected diseases.

Money, money, money
As is often the case, finding funding is a major challenge for individual researchers, which CEND aims to address. "In part, I think CEND is trying to help raise funds so faculty don't have to spend time on the administrative part of global health research," says Madon. She also sees a role for herself in helping campus researchers with research proposals to the NIH and other agencies and simultaneously "making clear to NIH that there is an interest in neglected tropical diseases and that UC Berkeley has a program in it and that we'd like to see enhanced funding [for] it."

For now, CEND is funded by a private donor. The money supports students like those from IIT in India who came to UC Berkeley this past summer, and students like Lisa Prach, who has gone to South Africa for her research. Madon describes other fundraising efforts: "We're writing one for minority and health disparities international training to send minorities and underrepresented students here to developing country sites of our collaborators. We're also drafting a proposal that could create an infectious disease modeling center on campus. We're trying to bring new resources to campus to help people engage in the kind of research that they want to, when there isn't a lot of funding [available]."

Is Big Science the future of drug discovery?
Big pharmaceutical companies have traditionally developed new drugs for diseases. They have large research programs, larger budgets and vast resources to throw at problems. However, drugs for neglected diseases, like trypanosomiasis, tuberculosis, malaria, and AIDS, challenge this time-tested model for discovery, simply because the people who suffer from these diseases cannot afford Big Pharma prices for their medicines. It has become increasingly clear that a new approach to the problem is needed, one that addresses the massive cost of discovering a new drug. Directing existing research efforts towards drug discovery may be the answer. This could be an advantageous situation for everyone— researchers have new, exciting, and socially relevant problems to work on, some of the best minds in the business are turned on to the most difficult problems, and drugs could be discovered at a far lower cost. CEND is a venture designed for this new kind of drug discovery and symbolizes so much of what is quintessentially Berkeley—world-class research with a social conscience.

Niranjana Nagarajan is a postdoctoral fellow in molecular and cell biology.

Want to know more?
CEND's website: globalhealth.berkeley.edu/cend/template.php?page=home

Welch lab: mcb.berkeley.edu/labs/welch

Alber lab: ucxray.berkeley.edu

World Health Organization site on neglected infectious diseases: http://www.who.int/features/factfiles/neglected_tropical_diseases/en/

New England Vampire Myths: ceev.net/biocultural.pdf

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