Finding of Long-Sought Drug Target Structure May Expedite Drug Discovery

ScienceDaily (Mar. 15, 2011) — Researchers have solved the three-dimensional structure of a key biological receptor. The finding has the potential to speed drug discovery in many areas, from arthritis to respiratory disorders to wound healing, because it enables chemists to better examine and design molecules for use in experimental drugs.

In this representation, the newly determined structure of the A2A receptor for adenosine is shown surrounding its synthetic agonist, a molecule that turns on the receptor. The spiral shapes and the slim connecting loops represent the receptor protein, winding back and forth through the cell membrane. The central (red) part of the agonist is critical for activation of the receptor. The top (tan-colored) part of the agonist, facing the outside of the cell, acts like arms to fill much of the remaining space in the binding site and stabilize the receptor, in order to obtain a crystallized structure. 

The researchers are from the National Institutes of Health, collaborating with labs at The Scripps Research Institute and the University of California, San Diego. The finding is published in the March 10 edition of Science Express.

"This is an important step forward -- it was impossible until recently to know how this type of receptor is switched on by chemical signals like a tiny machine," said Dr. Kenneth A. Jacobson, chief of the Laboratory of Bioorganic Chemistry in NIH's National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and an author on the paper. "The architecture of the activated receptor allows us to think in more detailed terms about the other half of the drug interaction. We hope that we're on the verge of a revolution that will expedite the process of crafting new drugs to treat disease."

With this finding, scientists in Jacobson's lab, including co-author Dr. Zhan-Guo Gao, will next work on testing this drug-engineering approach with similar molecules they have newly synthesized.

Jacobson and Gao are part of the NIDDK's intramural program, which enables basic scientists and clinicians of diverse skills and expertise to collaborate on solutions to some of the most difficult issues of human health. Several compounds from Jacobson's lab are currently in clinical trials as potential treatments for conditions including chronic hepatitis C, psoriasis and rheumatoid arthritis.

"Discoveries like this, with the potential to lead to future treatments in a wide variety of areas, are why NIH funds basic science," said NIDDK Director Dr. Griffin P. Rodgers. "By understanding the body at its smallest components, we can learn how to improve whole-body health."

A receptor is a protein that receives and sends signals to other molecules. The three-dimensional structure of the solved receptor also contains an agonist -- a chemical command signal from outside the cell -- in this case, an adenosine molecule. Similar to the function of a telephone receiver, the receptor acts as a sensor, picking up the message from the agonist and transmitting its information, which begins processes inside the cell.

The researchers discovered that a previously known agonist molecule would bind to its receptor target in a way that stabilizes the protein for crystallization. Once crystallized, the structure can be seen by bombarding it with X-rays. The agonist solidifies the protein by connecting to multiple parts of the receptor with its molecular arms, in the process initiating the function of the entire structure. This adenosine receptor, called A2A, counteracts inflammation and responds to organs in distress. It belongs to the G-protein coupled receptor family, which is involved in processes necessary for many drugs currently in use to take effect. These findings may lead to new drugs for many diseases.

The research was also supported by the National Cancer Institute and the National Institute of General Medical Sciences, both components of the NIH.

"Long-term NIH technology investments in structural biology, including the Protein Structure Initiative, have brought diverse teams of investigators together and yielded powerful methods like the ones used in this study," said NIGMS Director Dr. Jeremy M. Berg. "Receptors must undergo substantial changes in shape in order to function, and revealing these molecular dances in such great detail is an impressive accomplishment."

The NIDDK, a component of the National Institutes of Health (NIH), conducts and supports research on diabetes and other endocrine and metabolic diseases; digestive diseases, nutrition and obesity; and kidney, urologic and hematologic diseases. Spanning the full spectrum of medicine and afflicting people of all ages and ethnic groups, these diseases encompass some of the most common, severe and disabling conditions affecting Americans.

Single Gene Defect Causes Brain Tumor

ScienceDaily (Mar. 15, 2011) — Pilocytic astrocytoma, the most common brain tumor in children, is usually slow-growing and benign. However, surgeons often cannot completely remove the diffusely growing tumor. This means that patients need further treatment in order to destroy remaining tumor tissue. Chemotherapy or radiation therapy can lead to severe side-effects and have only little effect on these slowly growing tumors. Affected children therefore urgently need new, targeted therapies.

Tissue section of a mouse brain with a pilocytic astrocytoma. The brown staining indicates astrocytes. 

A typical genetic defect in these brain tumors is already known: "From our own research we know that there is a defect in the BRAF gene in the great majority of pilocytic astrocytomas," says Professor Dr. Peter Lichter of the German Cancer Research Center. This defect causes a cellular signaling pathway, which in healthy cells is active only in case of acute need, to be permanently activated.

Jan Gronych from Lichter's department has now studied, jointly with colleagues of Heidelberg University Hospitals, the actual relevance of the BRAF defect for carcinogenesis. To this end, the investigators packed a defective BRAF gene into a virus and thus introduced it into neuronal precursor cells of mice. In 91 percent of animals thus treated, tumors developed around the injection site. These tumors corresponded to pilocytic astrocytoma in terms of their biology, growth characteristics and tissue structure.

Cells of these tumors all showed the typical symptom of a defective BRAF gene: a permanently activated MAP kinase enzyme. "This proves that a single gene defect is really sufficient to cause pilocytic astrocytoma," said Lichter, summarizing the results.

A permanently active MAP kinase constantly transmits growth signals in cancer cells, while it is also their Achilles' heel: In recent years, a number of drugs have been developed which inhibit the enzyme activity of kinases very specifically and, thus, can impede cancer growth. The Heidelberg researchers have shown that brain cells which are driven to permanent abnormal cell division by a defective BRAF gene slowed down growth after treatment with kinase inhibitor sorafenib.

"Up to now, we did not have a suitable model system for testing newly developed drugs against pilocytic astrocytoma," says Peter Lichter. "The BRAF mice open up the possibility to test new kinase inhibitors or other drugs specifically for their effectiveness against pilocytic astrocytoma."

New Laser Technique Opens Doors for Drug Discovery

ScienceDaily (Mar. 15, 2011) — Researchers have demonstrated that a new laser technique can be used to measure the interactions between proteins tangled in a cell's membrane and a variety of other biological molecules. These extremely difficult measurements can aid the process of drug discovery.

Patterns created by the red laser in the backscattering interferometer.

Scientists estimate that about 30 percent of the 7,000 proteins in a human cell reside in the cell's membrane, and that these membrane proteins initiate 60 to 70 percent of the signals that control the operation of the cell's molecular machinery. As a result, about half of the drugs currently on the market target membrane proteins.

Despite their importance, they are difficult to study. Individual membrane proteins are extremely hard to purify, so scientists have very little structural information about them. In addition, existing methods to measure their activity have serious limitations. Most existing assays remove the membranes from their natural environment or modify them in a variety of different ways, such as attaching fluorescent labels, in order to analyze membrane protein activity.

"In addition to being expensive and time-consuming, these modifications can affect the target membrane's function in unpredictable ways," said Vanderbilt Professor of Chemistry Darryl Bornhop, who developed the new technique.

By contrast, in an article published online in the journal Nature Biotechnology, Bornhop's research group and their collaborators at The Scripps Research Institute report that the laser-based technique, called backscattering interferometry (BSI), can precisely measure the binding force between membrane proteins and both large and small molecules in a natural environment.

This is a powerful tool and a major advance in measuring membrane protein interactions," said Lawrence Marnett, director of the Vanderbilt Institute of Chemical Biology. "This is a powerful tool and a major advance in measuring membrane protein interactions," said Lawrence Marnett, director of the Vanderbilt Institute of Chemical Biology. Marnett, who is also Mary Geddes Stahlman Professor of Cancer Research, was not involved in the study but is planning on collaborating with the Bornhop group.

Lasers aid measurement

BSI is deceptively simple. It measures the binding force between two molecules mixed in a microscopic liquid-filled chamber by shining a red laser like those used in barcode scanners through it. When the geometry of the chamber is correct, the laser produces an interference pattern that is very sensitive to what the molecules are doing. If the molecules begin sticking together, for example, the pattern begins to shift.

In the new study, the researchers created synthetic membranes that contained a small protein, called GM1, that is a primary target that cholera toxins bind with in order to get into a cell. When they mixed these membranes with cholera toxin B, they measured a binding force consistent with that obtained by other methods.

The researchers performed similar validation tests with naturally derived membranes and three membrane proteins, one associated with breast cancer, another associated with pain and inflammation and the neurotransmitter GABA known to aid in relaxation and sleep and to regulate anxiety.

When they mixed the membranes containing each of these proteins with molecules known to bind with them, the BSI technique provided measurements that agreed with the values obtained by other methods, the scientists reported.

Vanderbilt has applied for and received three patents on the process and has several other patents pending.

The university has issued an exclusive license to develop the technology to Molecular Sensing, Inc. Bornhop is one of the founders of the start-up and serves as its chief scientist.

Vanderbilt research associate Amanda Kussrow and Michael Baksh, Mauro Mileni and M.G. Finn from The Scripps Research Institute contributed to the study, which was funded by awards from the National Institutes of Health, Joint Center for Innovative Membrane Protein Technologies and the Skaggs Institute for Chemical Biology.

New Role for an Old Molecule: Protecting the Brain from Epileptic Seizures

ScienceDaily (Mar. 15, 2011) — For years brain scientists have puzzled over the shadowy role played by the molecule putrescine, which always seems to be present in the brain following an epileptic seizure, but without a clear indication whether it was there to exacerbate brain damage that follows a seizure or protect the brain from it. A new Brown University study unmasks the molecule as squarely on the side of good: It seems to protect against seizures hours later.

Like putrescine in tadpoles The neurochemical putrescine surges in the brain after a seizure. By studying putrescine in tadpoles, researchers found that it exerts a calming effect, protecting the brain for a while against a second seizure.

Putrescine is one in a family of molecules called "polyamines" that are present throughout the body to mediate crucial functions such as cell division. Why they surge in the brain after seizures isn't understood. In a lengthy set of experiments, Brown neuroscientists meticulously traced their activity in the brains of seizure-laden tadpoles. What they found is that putrescine ultimately converts into the neurotransmitter GABA, which is known to calm brain activity. When they caused a seizure in the tadpoles, they found that the putrescine produced in a first wave of seizures helped tadpoles hold out longer against a second wave of induced seizures.

Carlos Aizenman, assistant professor of neuroscience and senior author of a study published in the journal Nature Neuroscience, said further research could ultimately produce a drug that targets the process, potentially helping young children with epilepsy. Tadpoles and toddlers aren't much alike, but this basic aspect of their brain chemistry is.

"Overall, the findings presented in this study may have important therapeutic implications," Aizenman and co-authors wrote. "We describe a novel role for polyamine metabolism that results in a protective effect on seizures induced in developing animals."

Detective work

The result that "priming" the tadpoles with a seizure led to them being 25 percent more resistant to a subsequent seizure four hours later was "puzzling," said Aizenman, who is affiliated with the Brown Institute for Brain Science. It took about a dozen more experiments before his team, led by graduate student Mark Bell, could solve the mystery.

First they hindered polyamine synthesis altogether and found that not only did the protection against seizures disappear, but it also left the tadpoles even more vulnerable to seizures. Then they interrupted the conversion of putrescine into other polyamines and found that this step enhanced the protection, indicating that putrescine was the beneficial member of the family.

Going with those results, they administered putrescine directly to the tadpoles and found that it took 65 percent longer to induce a seizure than in tadpoles that didn't get a dose of putrescine.

Further experiments showed that the protective effect occurs after putrescine is metabolized, with at least one intermediary step, into GABA, and GABA receptors are activated in brain cells.

"Potentially by manipulating this pathway we may be able to harness an ongoing protective effect against seizures," Aizenman said. "However I should caution that this is basic research and it is premature to predict how well this would translate into the clinic."

In the meantime, the research may also help explain a bit more about young brains in general, Aizenman said.

"Our findings may also tell us how normal brains, especially developing brains, may regulate their overall levels of activity and maybe keep a type of regulatory check on brain activity levels," he said.

In addition to Aizenman and Bell, the paper's other authors are undergraduates James Belarde and Hannah Johnson. The American Heart Association and the National Institutes of Health funded the study, while individual researchers were supported by the National Science Foundation, the Klingenstein Fund, and the Brain Science Siravo Awards for Epilepsy Research.

Lung Cancer Metastasis: Researchers Find Key Component -- And Possible Way to Block It

ScienceDaily (Mar. 15, 2011) — Researchers discovered a new, key component in the spread of lung cancer as well as a likely way to block it with drugs now in clinical trial.

The study was published in theJournal of Clinical Investigation.

A team led by scientists at The University of Texas MD Anderson Cancer Center found a way to identify metastasis-prone lung cancer cells and then uncovered a mechanism that shifts primary tumor cells into a more deadly type of cell with the capacity to move elsewhere in the body.

"We think tumors have to learn how to metastasize because they can't do it initially," said paper senior author Jonathan Kurie, M.D., professor in MD Anderson's Department of Thoracic/Head and Neck Medical Oncology. "Cells change in response to cues from their external environment."

About 90 percent of all cancer deaths are caused by metastasis -- the spread to, and invasion of, other organs. Lung cancer is the leading cause of cancer-related death in the United States, accounting for more than 157,000 deaths annually. The median five-year survival rate is 3.5%

Jagged2 silences protective microRNA

The researchers found that when a protein called Jagged2 binds externally to Notch, a membrane protein that sticks out through the surface of a cell, it suppresses a microRNA that thwarts metastasis inside the cell.

"Jagged2 suppresses miR-200 and drives metastasis as a consequence." Kurie said. "It's been known for some time that Notch is involved in cancer, but no one really knew how."

Two Notch inhibitors are in clinical trial at MD Anderson. "These drugs might suppress the ability of primary tumors to metastasize," Kurie said.

"One question is who is supposed to get these drugs," Kurie said. "Our data suggest that low levels of miR-200 may indicate a tumor's susceptibility to Notch inhibitors."

Jailing tumor cells

While the drugs don't kill a primary tumor, they do "keep the primary lung tumors in jail," holding them in place and blocking their transition to mobile cells, Kurie said.

This transition, from immobile epithelial cells, which line or cover an organ, to a migratory cell with the properties of a mesenchymal cell, is an early event in metastasis. Kurie and colleagues previously showed that miR-200 blocks this epithelial-to-mesenchymal transition. About 80 percent of all cancers begin in the epithelial cells of organs.

Telltale surface protein identifies metastatic cells

The first crucial research step was to identify and study non-small cell lung cancer cells prone to metastasizing.

Yanan Yang, Ph.D., study first author and a postdoctoral fellow in Kurie's lab, studied lung cancer cells in mice, searching for markers of metastasis. He homed in on a surface protein called CD133.

"In primary lung tumors, CD133 cells are under 1 percent of cells," Yang said. "In metastatic lesions, more than 80 percent of the cells have CD133."

Follow up studies determined that CD133-expressing cells were located on the perimeter of tumors, ideally situated for metastasis. More than half of mice injected with CD133-positive lung cancer cells had metastatic cancer, compared to less than 20 percent of those injected with CD133-negative cells.

Intensive study of CD133 metastatic cells revealed that they highly expressed Notch ligands (proteins that bind to specific receptors on other cells). Yang said they separately depleted two Notch ligands -- Jagged1 and Jagged2 -- from tumor cells. Removing Jagged1 had no effect, but cells with little Jagged2 did not metastasize.

"Because epithelial-to-mesenchymal transition (EMT) is a very early step in metastasis," Yang said, "we thought Jagged2 might regulate EMT." When they knocked down Jagged2 again, they found levels of the EMT-stifling miR-200 increased.

External signals drive change

Additional research found that Jagged2 reduced miR-200 by tipping a delicate balance between the microRNA and a protein called GATA3, which inhibit one another. Stimulating production of more GATA3 reduced levels of miR-200.

"Jagged2 increases the levels of GATA3, which in turn binds to the promoter of miR-200 and suppresses production of miR-200," Yang said.

"The study is among the first to show that mir-200 is regulated by specific signals emanating from the environment surrounding cancer cells. These signals are keys to understanding metastasis," Yang said.

One surprise, Kurie said, is that GATA proteins had been thought to suppress tumors. "In this case it's exactly the opposite."

The next step is to determine which of the four known Notch receptors suppress miR-200 and promote metastasis. The drugs currently under study are designed to inhibit an enzyme that cleaves and activates all Notch receptors. Drugs that target specific Notch receptors might be more effective inhibitors of metastasis, Kurie said.

Funding for the project was provided by grants from the National Cancer Institute, MD Anderson's Lung Cancer Specialized Program in Research Excellence grant from NCI, the David M. Sather Memorial Fund, The Armour Family Lung Cancer Research Fund, the Dan L. Duncan Cancer Center at Baylor College of Medicine, The ASCO Cancer Foundation, and the International Association for the Study of Lung Cancer.

Co-authors with Yang and Kurie were Young-Ho Ahn, Don L. Gibbons, Yi Zang, Wei Lin, Nishan Thilaganthan, Cristina A. Alvarez, and Daniel C. Moreira, of MD Anderson's Department of Thoracic/Head and Neck Medical Oncology; Moreira also is with the Medical School, Tecnológico de Monterrey, Monterrey, Mexico; Chad J. Creighton, Dan L. Duncan Cancer Center, Baylor College of Medicine; and Philip A. Gregory and Gregory J. Goodall of the Centre for Cancer Biology, Hanson Institute, Adelaide, South Australia, Australia, and Discipline of Medicine, University of Adelaide, Adelaide, South Australia, Australia.