Tools to identify cell signaling mechanisms yield better targets and drug leads with high binding affinity and specificity for previously untargeted diseases
Lori Valigra
Valigra is a freelance writer based in Cambridge, Mass.
Cell signaling plays a critical role in cell function, controlling everything from activating normal growth to spreading diseases. The mechanisms that transmit biological information from cell to cell thus make natural targets for new drugs designed to disrupt or alter the signals in an effort to treat disease. By honing traditional and new tools and techniques to more accurately identify cell signal targets, pharmaceutical and biotechnology companies are beginning to develop compounds with stronger binding affinity and very high specificity for many diseases, including difficult to treat ones like macular degeneration and psoriasis. In addition, scientists are finding they can leverage their drug discovery efforts by applying compounds that have a structure similar to their lead compound to other disease targets. For example, a compound for psoriasis can have a similar three-dimensional structure to another protein that could target bacteria.
 (Source: Archemix Corp.) |
Whether veins in the eye
One company that used a newer and somewhat unique tool is Eyetech Pharmaceuticals Inc., New York, a biopharmaceutical company that specializes in eye treatments. Last December, it received US Food and Drug Administration (FDA) approval for Macugen, its first drug. Macugen (pegaptanib sodium injection) is designed to treat neovascular, or “wet,” age-related macular degeneration, an eye disease that destroys vision in older people. (Partner Pfizer Inc. will market the drug.) Eyetech says Macugen is the first in a new class of drugs to target vascular endothelial growth factor (VEGF). The new drug is a pegylated anti-VEGF aptamer, a single strand of nucleic acid that binds to the VEGF 165 protein, the specific type of VEGF that signals the growth of the abnormal new blood vessel. In the wet form of the disease, those new vessels leak blood and cause vision loss or even blindness. Macugen acts as an antagonist and blocks the binding of VEGF to its receptor.
Some 15 million people in the United States have some form of age-related macular degeneration, and more than 1.6 million have the more advanced wet form. Each year more than half a million people worldwide lose their sight because of wet macular degeneration, and the number is expected to rise as baby boomers age. Existing treatments target a subgroup of wet macular degeneration accounting for 25% of the total sufferers. Macugen can be used for all types of wet macular degeneration, and is not limited to the size of the area of the vessel growth or the subtype of lesion.
“Macugen is the first anti-angiogenic treatment approved in ophthalomology,” M. Judah Folkman, MD, said in a statement when the drug was approved. Folkman, a professor at Harvard Medical School, made key discoveries on the mechanism of angiogenesis. “The anti-angiogenic approach specifically addresses . . . an underlying cause of blindness in age-related macular degeneration.” The drug received priority review under the FDA’s rolling submission-pilot I program using data from Eyetech’s phase II/III randomized, multicenter, double-masked clinical trials.
“We saw it was very well tolerated in the eye, and we decided for our subsequent drugs to use aptamers as our first choice of a drug platform,” says Tony Adamis, MD, who worked in the Folkman lab before cofounding Eyetech. Aptamers work extracellularly, so they don’t require entry into the cell to have a drug effect, which makes them different from siRNA and antisense methods. Adamis says aptamers bind to the protein target of interest with high affinity: Macugen has a picomolar binding affinity of 200 kD, and it has very high specificity. It binds to just the one isoform of VEGF and acts like an antibody, but it is a 28-mer oligonucleotide. In the future, there may be an extended-release formulation of Macugen. In addition, Eyetech plans to develop aptamers for other blinding diseases for which there are no good treatments, possibly including diabetic retinopathy.
 click the image to enlarge Macugen inhibits the binding of 125I-VEGF to VEGF receptors Flt-1 and KDR expressed on porcine aortic endothelial cells. (Source: SomaLogic Inc.) |
Selex and aptamers
Macugen is not only the first anti-angiogenesis drug approved for ophthalmology; it is also the first aptamer treatment approved by the FDA. Aptamers are made with a tool or methodology called Selex, or systematic evolution of ligands by exponential enrichment (see sidebar). Selex involves generating a large library of nucleic acid molecules, as many as 1015 different compounds, each of which contains a unique nucleotide sequence that can adopt a unique three-dimensional shape. A few of those molecules, the aptamers, have a surface that complements the target molecule.
The Selex technique was developed by Larry Gold, PhD, and graduate student Craig Tuerk, PhD, in Gold’s lab at the University of Colorado, Boulder, in 1988. “My graduate student did an experiment studying an RNA molecule, and we did the first selection of aptamers. Instead of getting back one sequence, we got back two, and the two were so different from each other, but they each bound to the same protein. We were shocked,” says Gold. “We realized we could make molecules using Selex. It was the single most thrilling scientific moment of my life.”
Gold licensed the Selex technology from the university and started a company called NeXstar Pharmaceuticals Inc., Boulder, Colo., in 1991 to further develop the technology. By early 1998, NeXstar had taken a compound called NX 1838, later to be renamed Macugen, into early clinical development. “Selex technology is essentially the generation of a randomized nucleic acid library, and the process involves doing successive rounds of affinity selection,” says Nabojsa Janjic, PhD, a NeXstar scientist who spearheaded the early work with VEGF and Selex. Janjic is now senior vice president of R&D at Replidyne Inc., Louisville, Colo. “This basically allows one to identify nucleic acids that bind with very high affinity to a particular molecular target.”
Eyetech signed on Archemix to make the Macugen aptamer using the SELEX process. Adamis says the first step was placing a pool of individual nucleotides in a test tube with the appropriate RNA or DNA polymerase to make aptamers that were poured over a column of VEGF to determine which ones would bind with fairly high affinity. Those binders were amplified, and then more were made to find the ones with the highest affinity. Several cycles were repeated until the best binders were discovered. The end result was the Macugen aptamer. Eyetech then tested the aptamer in its models to ensure that it behaved in the expected way, that is, to stunt vessel growth. It also performed standard toxicology and pharmacology tests.
Aptamer disadvantages
There are some disadvantages to aptamers. They typically are aimed only at extracellular targets, either cytokines like VEGF or the extracellular domains of a membrane protein. “You can’t easily get aptamers inside cells. They are too big and have a lot of charge on them,” says SomaLogic’s Gold. Cost and stability also have been issues. The cost of an aptamer was about $100,000 per gram in 1990, but the price is dropping. “We now have projections on a commercial scale that we will be making our lead aptamer at a cost of $25 to $30 per gram,” says Marty Stanton, PhD, executive vice president of Archemix.
Stanton adds that in the last decade, methods have been developed to stabilize the aptamers by modifying DNA and RNA and attaching a PEG. For example, certain places on DNA make it susceptible to degradation, but those locations can be stabilized by making derivatives of them. “So between building the molecule’s drug-like properties and being able to make them at competitive costs, I think this makes them commercially viable, and we’re likely to see more aptamers.”
Stanton says that aptamer development is appealing because it is comparatively fast. “We have been able to go from initiation of a program into the clinic in a total of 36 months. In the development of aptamer therapeutics, the initial discovery is far faster than any other class of molecules because these things have predictable pharmacology.” In addition, Aptamers are chemically synthesized. “Unlike antibodies, which require $300 million fermentation plants, these can be made in a suite that’s not much bigger than my office, which is small,” Adamis says. “It’s a straightforward chemical synthesis, much like small molecules.” Archemix has a new aptamer in phase I development called ARC183 in collaboration with Nuvelo Inc., Sunnyvale, Calif. It is an anticoagulant for potential use in coronary artery bypass graft surgery.
| Anatomy of an Aptamer University of Colorado professor Larry Gold’s discovery of the Selex process to identify high-affinity aptamers to be used as drugs was an awe-inspiring moment, says Marty Stanton, PhD, executive vice president of Archemix Corp., Cambridge, Mass. “Until 20 years ago, people thought DNA and RNA carried information in the cell,” Stanton says. “But what was discovered is that DNA and RNA also can fold up into a three-dimensional structure and can do everything a protein can do. That was a staggering observation.” He says Gold determined the Selex method to synthesize a very large number of different RNAs or DNAs and find the one that would fold up just right so that it binds to a protein or other molecule. “Over the last 10 years we’ve found you can make aptamers that bind to a wide variety of different proteins and are able to modulate the properties of that protein so you can make aptamers that are drugs,” he says. “The Selex process has become very robust to discover aptamers in a matter of weeks or months.” |
Stanton sees other uses for aptamers in drug discovery and development, including in target validation. His company has a partnership funded by Johnson & Johnson Pharmaceutical Research and Development, Beerse, Belgium, to validate G-protein-coupled receptor targets. Aptamers also could be used, he says, to do protein expression profiling for drug discovery or immunohistochemistry, ELISA-like assays, Western blots (using aptamers instead of antibodies) and affinity chromatography. They also potentially could be put onto viral or naked DNA vectors to knock out protein function inside of cells.
Universities have also been working on aptamers, even though Archemix and SomaLogic own most of the rights to use and license Selex and aptamer development. Stanton says the two companies plan to soon announce a university licensing program that will provide rights to universities at no cost. “We want to make it explicitly clear that we are allowing them to do this research.”
The right stuff
For W. Michael Gallatin, PhD, vice president and scientific director at ICOS Corp., Bothell, Wash., developing a drug for psoriasis was a matter of getting a compound that was potent enough and that blocked the correct stage in the progression of the disease. The company had an LFA-1 antagonist in phase II clinical trials, but pulled it back after deciding it needed to redefine those parameters.
LFA-1 is an integrin, or a heterodimeric cell surface adhesion molecule that is selectively expressed on leukocytes. It plays a major role in activating and trafficking T lymphocytes in the tissue where there is inflammation. In the initial trial, Gallatin says it was clear that about 80% of the cell surface LFA-1 needed to be saturated with an antagonist in order to block the movement of leukocytes across the blood vessel wall. The company succeeded in doing that in humans. But he says work published by Genentech Inc., South San Francisco, Calif., and others has since shown that treating psoriasis clinically is not necessarily a factor of the number of T cells present in the tissue lesions, but the state of activation of T cells once they are in the tissue.
“We had initially assumed that if you blocked traffic into the skin that you would have a positive impact on psoriasis,” he says. “But psoriasis patients probably have enough T lymphocytes already in the skin. You need much more potent compounds to block the activation step, the cell signaling step, than you need to simply block the traffic step,” says Gallatin.
Filed Under: Drug Discovery
