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Obtaining a high-resolution crystal structure of a protein, a receptor or an enzyme, for instance, has been at the forefront of the drug design field for many years. Finding small molecules that will dock with the active site of the protein and either stimulate it or inhibit it is the basis on which many pharmaceutical products were built and are thought to work. But, what if that fundamental concept were wrong? This is the sobering and at the same time very important conclusion made by researchers at Leiden University in The Netherlands and the Scripps Institute, La Jolla, California. Ad IJzerman, head of the division of medicinal chemistry at the Leiden/Amsterdam Center for Drug Research, and postdoctoral researcher Rob Lane have been working with La Jolla crystallographer Ray Stevens on elucidating the structure of a G-protein coupled receptor protein, the A2A adenosine receptor. This protein is one of the main targets in the body for the stimulant caffeine and is inextricably linked to Parkinson's disease. Researchers are keen to understand its precise role and to develop an accurate model that could be used to develop novel treatments for this debilitating disease. Of course, obtaining the crystal structure of this receptor, which is bound to the cell membrane should provide important insights. Moreover, if researchers can determine the crystal structure with and without a small molecule in the binding site, all the better. This is thought to be the best approach to improving our understanding of the mode of action of any putative drug that interacts with the receptor, and this is also considered a generally applicable approach for other protein targets. But, membrane-bound proteins are notoriously difficult to crystallise and without crystals there is no crystal structure. "For decades scientists from all over the world have struggled to get the crystal structure of this type of G protein-coupled receptor", IJzerman explains. "These arduous attempts are easily understood when one takes into account that the whole family of these proteins are the targets for almost half of the medicines that are available in the pharmacy shop. It seemed an impossible task, since these proteins are in the cell wall, which means they are in a fatty environment, and are fatty themselves. We all know that fat does not crystallize easily." The team at La Jolla believed they had found a rather elegant solution to this perennial problem. They coupled the receptor protein to another protein that they knew to be a fast crystallizer. This allowed them to obtain tiny crystals, using very advanced crystallization equipment, of the fusion product. The team found that this was sufficient to let them crack the structural code of the membrane protein in which they were really interested. With this information to hand the La Jolla team turned to their colleagues in The Netherlands. This specific type of receptor is at the heart of research in the division of medicinal chemistry of the Leiden/Amsterdam Center for Drug Research. By joining forces the two teams hoped to construct the biochemical characteristics of the receptor and so understand its pharmacology in much more detail than ever before. By the end of June 2008, the team had the first crystals of suitable quality. But, the crystal structure determination was to throw up a big surprise for the researchers that will have resounding implications for the whole pharmaceutical industry. "The binding site for drugs on this receptor is very different from the one that had been found on two other receptors for which we currently know the crystal structure", explains Lane. "In the adenosine A2A receptor a small molecule, prosaically called ZM241385, is co-crystallized. This compound has high affinity for the receptor, and therefore it is best described as some sort of 'supercaffeine', a type of molecule that we had worked on in Leiden before. Lane continues, "The drug is in a very different position than was expected on the basis of the other crystal structures. And there's the rub; almost everybody in the world of drug design has so far used receptor models that may not be so useful at all. That is the sobering and at the same time important discovery we made." IJzerman told SpectroscopyNOW about the long-term relevance of the work to the pharmaceutical industry. In the year 2000, the rhodopsin crystal structure was published, but the current research compares the binding sites for retinal in rhodopsin and ZM241385 in the adenosine A2A receptor. "They're very different with ZM located in a perpendicular orientation relative to retinal," IJzerman explains, "So far, many (academic and industrial) molecular modelers have taken the binding site of retinal in rhodopsin as the template to model other G protein-coupled receptors and their drug binding sites." He adds that, "Many such receptor models have been published in literature, and they may not be so useful after all, as the binding site in one of the first real, i.e. experimentally determined, GPCR structures is different already." So, what does this say about how countless drugs actually work. If they were designed with one aim in mind then presumably this undermines the concept of targeted drug design. IJzerman explains this away as the fact that most models are made in retrospect. "After the identification of an active lead or optimized lead structure, the model is made," he says, "The room for ligand binding in a receptor homology model is often ample enough as retinal itself is a relatively 'big small' molecule. Then it is easy to accommodate the ligand of choice into the receptor of choice. Visualization is often a convincing argument, even if it is wrong." Reference:
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