James Netterwald, PhD, MT(ASCP), Senior Editor
X-ray crystallography has been used in drug discovery since the 1980s. Recently it has enjoyed a resurgence.
Knowing the structure of a protein used to be of greater importance to basic biological science than to applied biomedical science. However, as these fields converge, knowing and understanding protein structure has become imperative for both. X-ray crystallography, the most important tool for determining protein structure, is the preeminent tool of the relatively new science of structural biology, but its use in science in general is not new at all.
“X-ray crystallography dates back to almost 100 years now. It was originally used by geologists to study the structure of minerals,” says Harren Jhoti, PhD, founder and
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chief scientific officer of Astex Therapeutics, Cambridge, UK. At 100 years and counting, the technology is still going strong. In fact, X-ray crystallography is not limited to academic structural biology anymore, but has become a major tool for drug discovery.
But when did X-ray crystallography enter the drug discovery picture, especially for proteins? “To me, the two founding efforts were Vertex and Aragon, which was around 1984 or 1989,” says Ray Stevens, PhD, professor of molecular biology and chemistry at The Scripps Research Institute, La Jolla, Calif. Then there was a long gap. “It wasn’t until the late 1990s, when Syrrx, Astex, and Structural Genomix were founded with new accelerated technologies, that there was whole new resurgence of structure-based efforts.”
How it works
The principles of X-ray crystallography dictate that after firing X-rays at matter, the X-rays will bounce (diffract) off of matter in a very ordered manner specific to that particular crystal. The ways in which X-rays are diffracted are governed by various laws of physics.
In order to determine the locations of atoms in a molecule, a tool with a resolving power high enough to determine the distance between two atoms is needed. “Any kind of radiation can be diffracted. It just so happens that X-rays are diffracted by the spacing between atoms and areas of high electron density in the crystal,” says Jeffrey Deschamps, PhD, research chemist for the Naval Research Laboratory, Washington, DC.
X-rays may have the resolving power, but they don’t amplify the diffraction pattern, especially with single molecules. This is why a crystal is needed.
The process of X-ray crystallography must, of course, start with the crystallization of a protein of interest. This procedure can take on many forms. For example, one could grow the crystal by mixing the protein with a variety of different chemicals or by slowly concentrating a solution of the protein.
The protein crystal can also take on a number of different conformations. But the crystal does not need to contain the active conformation. More often than not, the inactive form is the one that is predominantly isolated, says Rick Artis, PhD, vice president of lead generation, Plexxikon Inc., Berkeley, Calif. Furthermore, he points out that locking an enzyme into an inactivatable form can be useful from a therapeutic perspective. After making the crystal, there are many sources of X-rays to use for the analysis. “We exclusively use high energy synchrotron radiation at places like the Advanced Light Source at the University of California at Berkeley.”
In his position at the Naval Research Laboratory Deschamps does a lot of structural studies funded by National Institute on Drug Abuse and the National Institutes of Health. Deschamps provides them with information on chemical conductivity and absolute configuration. This illustrates the wide range of applications made possible by X-ray crystallography. Most importantly, in addition to being used to determine protein structure, X-ray crystallography can also be used to determine the three-dimensional structure of a drug itself. For example, X-ray crystallography is also used to identify drug molecule polymorphisms.
A drug discovery project used to start with a compound found through a high-throughput screen. Then, when an interesting protein target was discovered and a crystal of it prepared, a researcher would see whether they could soak the crystal with a solution of the compound, with the hope that the compound would end up going into the crystal. Plexxikon does things differently, says Artis. “We prefer to do a technique known as co-crystallization, where you crystallize, from a solution, the protein target and the compound of interest.”
However, the point at which X-ray crystallography comes into the drug discovery and development process depends on the purpose for which it is used and that, of course, depends on whom you ask. It can be used at the start, where it gives a researcher the advantage of knowing the three-dimensional structure of a drug target before testing drug compounds against it, says Jhoti. For example, “if you don’t have lead compounds for your target yet, knowing the three-dimensional structure of your protein can be helpful in the sense that it can allow you to use some computational chemistry tools to find early lead compounds.” Artis says it is more commonly used later in the drug discovery process, “partly because of the time it takes to get high-quality crystallography up and running for any given target. Historically, this was the case in many big pharm companies up until the last five years.”
Obstacles, challenges
Although X-ray protein crystallography is an extremely powerful tool, there are a few obstacles blocking its path to prominence. For example, “protein molecules tend to be a lot more fragile in the sense that they are not resistant to extreme heat or osmotic pressure, whereas smaller molecules, such as the drug compounds themselves, are [resistant],” says Jhoti. This makes proteins more difficult to crystallize.
Another major challenge to doing structure-based drug design is that it takes a longer time to crystallize a protein than to crystallize a smaller molecule. Glycosylation and
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oxidation of proteins can occur during protein synthesis and crystallization, respectively, consequently hindering crystallization. Researchers must also look out for protein degradation during crystallization, as it can affect the crystal’s structure and yield inaccurate data. The bottom line is that anything that hinders an X-ray crystallographic analysis delays the start of lead optimization in a structure-based drug design process.
But there are ways to make protein crystallization a lot easier. For example, one of the tricks used to facilitate protein crystallization is to vary the amino sequence of the protein, such that the new variant is still in a native conformation but is easier to crystallize. On the other hand, Artis suggests that one can avoid such a delay by choosing targets that crystallize well and quickly, therefore expediting the lead discovery process.
“If you had asked me about ten years ago, I would have said that the biggest challenge was crystallizing a protein,” says Stevens. However, “with a lot of the technology that has come out in the last five to seven years, the challenge now is finding a protein construct that will behave the best during crystallization.”
Because of the dual personality membrane proteins have, with both a hydrophobic and hydrophilic interface, the success rate for crystallization of membrane proteins is still very low. “This is why the NIH has a road map initiative where many scientists come together and decide what are the biggest challenges facing biomedical research. It was decided two years ago that membrane proteins are one of the biggest challenges and one of the most important technological barriers to overcome,” says Stevens. These meetings resulted in a significant increase in funding for membrane protein structural biology research.
There are many pros and cons to using X-ray crystallography in the drug discovery process. “The biggest pro is that the therapeutic quality of drug molecules increases with higher quality structural information,” says Stevens. Another major pro is that a chemist can use the tool to design drugs in a rational way. For example, Jhoti says, “if there is a metabolism problem with a drug, and you try to fix it by modifying the drug, the knowledge acquired through X-ray crystallography allows you to understand how the drug interacts with its target, so you can make a more informed decision as to which groups to modify.”
But, he adds, the downside is that “you cannot make the inference that a given protein’s structure is the same in a crystal as it is in a cell.” This is due to the fact that the biologically relevant structure(s) are difficult to determine crystallographically. So, for a given protein, assays for activity and structure are usually performed in parallel to ensure that the crystal obtained also contain the relevant bioactivity. Traditionally, X-ray crystallography took a long time to perform, with the structure of some targets taking several months to a year to deduce, says Stevens. The new technologies are changing all of that.
Finally, X-ray crystallography is expensive. This presents a major obstacle to smaller labs that don’t have the funding. In addition to equipment costs, there is also the cost of training staff members to use the equipment. “The companies who sell X-ray crystallographic equipment would like you to believe that any chemist could learn to use the equipment and produce crystal structures. That’s true if it’s a routine structure without any [technical] problems,” says Deschamps.
Getting an early start
The fact that X-ray crystallography allows one to determine the three-dimensional structure of a drug target has really had a positive impact on the process of lead discovery and optimization. According to Artis, X-ray crystallography was historically used late in the drug discovery process to get a snapshot of how a lead compound was interacting with its target. However, Plexxikon Inc. and other companies in the structure-based drug discovery field were built on the premise that obtaining structural information before any lead compound synthesis takes place allows the researcher to design compounds for lead optimization. In other words, X-ray crystallography now has predictive value.
“We internally have spent quite a lot of effort using modern computational techniques and various informatics approaches to be able to use the structure to look for chemistry,” says Artis. He adds that in about half of their efforts, they get a predictive model to guide downstream chemistry qualitatively as well as quantitatively.
This brings to light an emerging trend: Most companies are using X-ray crystallography at a much earlier stage than before. Artis says that now, when a company identifies a target they would like to screen using a high-throughput biochemical assay, they are more likely to also perform a crystallization screen of the drug-target complex at the same time.
There have been other improvements, too. “Because of advancements in the equipment and software, there are structures that we can solve today that we could not solve 15 years ago,” says Deschamps. Some of the improvements include the use of X-ray bulbs, which allow researchers to produce the X-ray particles in their own labs, and synchrotron radiation, which is produced in large facilities via a particle storage ring. Ethan Merritt, PhD, research associate professor at the University of Washington, Seattle, is among the many users of the Stanford Synchrotron radiation facility.
However, Merritt does not need to travel to Stanford to conduct his experiments in person. Instead, he sends the samples to the facility and then controls the experiment remotely from his home-base; this major improvement is due to the robotic operation of the X-ray system, an improvement made in the last year.
Because of vast improvements in X-ray crystallographic methods, researchers see X-ray crystallography continuing to play a role in drug discovery in the future. “I expect that [X-ray crystallography] will become routine in the future,” says Artis, who adds that this is because nearly every aspect of the drug discovery process has become automated, and that includes X-ray crystallography.
This article was published in Drug Discovery & Development magazine: Vol. 9, No. 11, November, 2006, pp. 36-40.
Filed Under: Drug Discovery
