05/03/93
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STANFORD - Researchers at the Stanford Synchrotron Radiation Laboratory (SSRL) are using synchrotron radiation to study the three- dimensional structure of HIV-protease, an essential enzyme in the multiplication of the virus that causes AIDS.
The project, a collaboration with the Palo Alto-based drug company Syntex, aims at designing a drug that would knock out the enzyme, thus preventing the virus from spreading to new cells in an infected person.
SSRL crystallographers Henry Bellamy, Hartmut Luecke and Mike Soltis are developing a high-resolution model of the atomic interface between HIV-protease and an inhibitory molecule, a potential drug developed by Syntex.
"Here at SSRL, we have crystallized the protease together with an inhibitory molecule and now want to know exactly how that inhibitor binds," Luecke said. "Once Syntex has that information, they may be able to modify this drug candidate to make it even more effective."
Having detailed structural information will help researchers who perfect a drug that mimics the enzyme's normal reaction partner -- a viral molecule to which HIV-protease would normally bind. A drug that looked just like that reaction partner could trick the protease into mistaking the drug for the real thing and sticking tightly to it. Sitting smack in the active site of the enzyme, the drug could paralyze HIV- protease.
While that rational drug-design effort could be a good starting point for clinical trials, an approved medicine still may be years away.
The three-dimensional computer image of HIV-protease results from a method that shoots X-rays through tiny protein crystals to find out their molecular structure. The process is called protein crystallography.
Thanks to the intensity of the synchrotron X-rays - extremely intense radiation that charged particles emit when bent on a circular path at near-relativistic speed - the SSRL crystallographers get a clearer picture of the area where the potential drug snuggles up against the protease than they could with standard crystallography equipment.
"Currently, our resolution is close to full atomic resolution, but soon we probably can do even better and get real atomic detail," Luecke said. "With more accurate distances between the individual atoms, we can judge the interactions between the inhibitory molecule and the enzyme much better."
Researchers had long thought that using such a powerful beam was not possible because it would destroy the crystal. However, Professor of Chemistry Keith Hodgson and his collaborators at SSRL have found that the synchrotron light allows them to collect all the necessary data before the crystal starts crumbling away.
To image HIV-protease, the researchers immerse a tiny crystal of the enzyme in the synchrotron X-rays. By gradually rotating the crystal, they capture many different viewpoints and imprint the outcoming waves on a screen. That information is invisible to the human eye, so a laser picks it up by scanning along the screen.
An image processing computer then comes up with the first round of data: many thousands of dots representing the diffraction pattern of the synchrotron light as it leaves the crystal. Like an artist's work, these dots are splashed on the computer screen in a beautiful pattern, but it does not yet look like a protein. Now the tedious work starts, because the scientists must analyze the intensity of every single dot separately.
A mathematical transformation yields the next round of computer data: long columns of numbers that translate the intensity of each dot into a place in space. After converting these numbers with a computer graphics program, a colorful, speckled protein-blob finally emerges. It represents a three-dimensional map of the electron distribution of the protease that enables the researcher to construct a model of the actual atoms and their bonds.
That last step may take most of the crystallographers' time and requires experience and old-fashioned human intuition, despite the extensive help of computers throughout the process.
"Fitting the map to a real protein is usually the hardest part, but after years of seeing proteins on the screen, you develop intuition. Sometimes you can tell what the protein must be like although you can't explain it to others," Luecke said.
In this project, constructing an atomic model of HIV-protease with the drug candidate tucked against it may be quite straightforward because other scientists have already solved related structures of the enzyme, which will help the SSRL researchers to figure out theirs.
While those structures are similar, they will not be identical to the one studied at SSRL since one enzyme usually comes in many slightly different shapes. It twists a bit when binding to a chemical and it also "breathes," wiggling subtly as it floats through the test tube. A crystal structure is but a snapshot of the vibrant enzyme.
The twisting and breathing goes along with incredibly tiny changes in energy. Because the protease bases the answer to the decisive question of 'to bind or not to bind' the inhibitor on these energy changes, they become a central problem in designing a new drug.
Such rational drug-design research will expand as more and more crystal structures of crucial disease proteins are being solved.
"Protein crystallography was an exclusively academic field until about 15 years ago," Bellamy said, "but the advances in protein cloning have helped turn it into a widely applied science."
Molecular geneticists now can supply theoretically any protein in large amounts for crystallographers to study its structure.
Protein cloning, while frequently raising public concern about the safety risks of genetic manipulation, has made the HIV-protease research less dangerous because the virus is no longer necessary to obtain the protease.
"Nobody likes working around large amounts of the AIDS virus, but producing and studying just one of its crucial proteins is a safe experiment," Bellamy said.
SSRL is supported by the U.S. Department of Energy and by the National Institutes of Health.
This story was written by Gabrielle Strobel, a science writing intern at the Stanford News Service
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