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The research emerged from the lab of Susan Lindquist, Ph.D., a professor of biology at the Massachusetts Institute of Technology (MIT), a member of the Whitehead Institute for Biomedical Research, and a Howard Hughes Medical Institute investigator. Dr. Lindquist is also an investigator at the Massachusetts General Hospital (MGH)/MIT Morris K. Udall Center for Excellence in Parkinson's Research, one of 14 such centers funded by NINDS to develop treatment breakthroughs for Parkinson's disease. The study received additional funding from NIH's National Institute of Environmental Health Sciences, and from the Michael J. Fox Foundation and the American Parkinson's Disease Association. Parkinson's disease attacks cells in a part of the brain responsible for motor control and coordination. As those neurons degenerate, the disease leads to progressive deterioration of motor function including involuntary shaking, slowed movement, stiffened muscles, and impaired balance. The neurons normally produce a chemical called dopamine. A synthetic precursor of dopamine called L-DOPA or drugs that mimic dopamine's action can provide symptomatic relief from Parkinson's disease. Unfortunately, these drugs lose much of their effectiveness in later stages of the disease, and there is currently no means to slow the disease's progressive course. In most cases, the cause of Parkinson's disease is unknown, but there are recent, tantalizing clues. Investigators have discovered that vulnerable brain cells in patients with Parkinson's disease accumulate a protein called alpha-synuclein. Moreover, genetic abnormalities in alpha-synuclein cause a rare familial form of the disease. Dr. Lindquist and her team previously showed that when yeast cells are engineered to produce large amounts of human alpha-synuclein, they die. In their new study, Dr. Lindquist and her team tested whether yeast could make cyclic peptides that would save them from alpha-synuclein's toxicity. Cyclic peptides are fragments of protein that connect end-to-end to form a circle. Although cyclic peptides are synthetic, they resemble structures that are found in natural proteins and protein-based drugs, including pain killers, antibiotics and immunosuppressants. Cyclic peptides that suppress alpha-synuclein toxicity could be candidate drugs for Parkinson's disease, or they could help researchers identify new drug targets for the disease. "Our technique, which capitalizes on a long line of investigation in my lab, will lead to a whole new way to obtain small molecule tools useful for improving our understanding of disease mechanisms and for developing new therapies," says Dr. Lindquist. She notes that her lab and others have modeled many human diseases in yeast and in other kinds of cells. Joshua Kritzer, Ph.D., a chemist and postdoctoral fellow in Dr. Lindquist's lab, designed and executed the cyclic peptide strategy. His procedure involves exposing yeast cells to short snippets of DNA that the cells can absorb and use to make cyclic peptides. Then, he flips the genetic switch that causes the cells to produce toxic levels of alpha-synuclein. If the yeast make cyclic peptides that suppress alpha-synuclein toxicity, they live; if not, they die. This simple assay enables testing millions of cyclic peptides simultaneously in millions of yeast cells. The process is extremely rapid and much less expensive compared to other techniques used to screen large number of chemicals with an eye toward new drugs. "We are making the yeast do a ton of work for us. They make the compounds and then they tell us which ones are functional," Dr. Kritzer says. Out of a library of 50 million cyclic peptides, only two saved the yeast from alpha-synuclein toxicity. Dr. Lindquist's team collaborated with other researchers to test these two cyclic peptides in C. elegans, a millimeter-long worm with a small number of dopamine-producing neurons that are easy to examine and count. Those neurons are vulnerable to alpha-synuclein toxicity, but they were less vulnerable and more likely to survive in worms that were genetically modified to make either of the two cyclic peptides. Guy Caldwell, Ph.D., and Kim Caldwell, Ph.D., professors of biology at the University of Alabama in Tuscaloosa developed this C. elegans model, and performed the testing. The researchers have not yet determined why the cyclic peptides are protective. They found that the cyclic peptides do not affect a system of transport inside cells known as vesicle trafficking – which was a surprise, since alpha-synuclein and other proteins that have been implicated in human Parkinson's disease are believed to play a role in vesicle trafficking. However, the researchers observed that the two peptides share a structure that may hold clues to their targets. "This protein structure has important biological functions," says Dr. Kritzer. It is found in a class of antioxidant proteins known as thioredoxins, in proteins that shuttle metals around a cell, and in proteins that regulate gene activity. The connection to antioxidants and to metals ties into other lines of research. NINDS is currently supporting clinical trials in patients to test whether specific antioxidants slow the progression of Parkinson's disease. High doses of heavy metals such as lead, manganese, iron and mercury are known to be toxic to brain cells. The researchers are conducting further experiments to explore how cyclic peptides prevent cell death. They are also adapting their system for making cyclic peptides so that it can be used in other cell types (including human cells) and other diseases.
Press release of the Whitehead Institute for Biomedical Research:
New method may accelerate drug discovery for difficult diseases like Parkinson'sCAMBRIDGE, Mass. - Whitehead Institute scientists have developed a rapid, inexpensive drug-screening method that could be used to target diseases that until now have stymied drug developers, such as Parkinson's disease. This technique uses baker's yeast to synthesize and screen the molecules, cutting target discovery and preliminary testing time to a matter of weeks. The current drug discovery process is arduous, requiring identification of potential drug targets, synthesis of large collections of molecular compounds that might interact effectively with an identified target, screening of compounds with expensive assays and robotics, and defining the compounds' structures largely through trial and error. At the end of this months-long process, a large team of chemists and biologists usually deem only 1% or fewer of the compounds worthy of further testing in living cells. A novel method, demonstrated by Whitehead scientists and described in the July 13 issue of Nature Chemical Biology, uses baker's yeast cells to perform most of the same work in a matter of weeks, with the added benefit that the testing is all done in living cells. At the core of this approach are extremely small proteins, called cyclic peptides, which are capable of targeting the protein-protein interactions found in almost every cellular process. Most current drugs act by wedging themselves into small pockets on the surfaces of target proteins. However, these traditional drugs are unable to adhere to smooth, flat protein surfaces, rendering the drugs ineffective for inhibiting the key interactions among proteins that occur at these surfaces. Cyclic peptides have the ability to bind where traditional drugs cannot, allowing for the identification of previously overlooked targets to fight disease. "We're getting at a chemical space that is very underexplored by traditional drug development and screening," says Joshua Kritzer, author of the Nature Chemical Biology paper and a postdoctoral researcher in Whitehead Member Susan Lindquist's lab. "I think it's a very exciting method," says Lindquist, who is also a professor of biology at MIT and a Howard Hughes Medical Institute Investigator. "It provides much greater diversity in the chemical compounds you can study because you can screen millions of compounds in the same go." Adapting previous work by the Benkovic lab at Pennsylvania State University, Kritzer created a vast "library" of cyclic peptides containing various amino acid combinations. He then inserted the cyclic peptides into cells of a well-established yeast model of Parkinson's disease that was created in the Lindquist lab. Parkinson's disease is a neurodegenerative disorder characterized by tremors, muscle rigidity, and slowed movements. In the neural cells of Parkinson's patients' brains, researchers have noted Lewy bodies, abnormal aggregates primarily composed of the protein alpha-synuclein. There is currently no cure for the disease, and current Parkinson's therapies only address disease symptoms. In the Lindquist yeast model, the cells exhibit many of the hallmarks of cells in Parkinson's disease patients' brains, including death due to toxic overproduction of alpha-synuclein. Once the cyclic peptides were inserted into the model yeast cells, Kritzer switched the yeast into Parkinson's mode and waited to see which yeast cells survived. Of the approximately 5 million yeast cells that were inserted with a cyclic peptide, Kritzer ended up with only two cyclic peptides able to rescue the cells from death. After sequencing them, Kritzer found that both effective cyclic peptides needed only the first four amino acids to work and those amino acids had a common motif (cysteine – any amino acid – a hydrophobic amino acid – cysteine). This particular four-amino-acid motif is very similar to some important biochemical structures, including molecules that oxidize or reduce other molecules and molecules that bind to metals. Interestingly, there are already links between Parkinson's and the metal manganese. Overexposure to the metal manganese can lead to parkinsonism, a Parkinson's disease-like syndrome. Also, earlier work conducted by Aaron Gitler and Melissa Geddie in the Lindquist lab found that the normal version of the gene PARK9, which can be mutated in Parkinson's disease patients, protects cells from toxic levels of manganese. With these possible modes of action in mind, Kritzer and colleagues are now trying to figure out how the new cyclic peptides work. Using the Lindquist yeast model and a worm model of Parkinson's disease from the Caldwell lab at the University of Alabama, they confirmed that the effective cyclic peptides have the same potency as natural genes that regulate Parkinson's related cellular processes, but intercept the disease's progress at a later point. This demonstrates that these cyclic peptides act at a point in the disease process that had not been targeted by other, more traditional approaches. According to Kritzer, who will be starting this September as an Assistant Professor of Chemistry at Tufts University, a next step in this line of research will be to determine precisely how the effective cyclic peptides affect Parkinson's disease cells – by changing reduction or oxidation within the cell, binding to metal molecules, or perhaps another mechanism. In addition, more potent structures may be possible, so the cyclic peptides' known structure can be used as a starting point for more libraries which may produce even more effective versions. Lindquist also says the technique is not limited to just yeast or just Parkinson's disease. "There's absolutely no reason we couldn't apply the same process to mammalian cells. And it should be applicable to all sorts of diseases that are modeled in yeast," she says. "In fact, that's some of the stuff we've started doing with this technique."
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