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June 23, 1999

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Target for Cystic Fibrosis Drugs Found

Biochemists Unravel CF at Most Basic Level

In an advance that promises to speed development of new drugs for cystic fibrosis, Johns Hopkins biochemists have discovered what goes awry inside the cells of CF patients at the most basic level.

It's a folding problem that might be familiar to any home-maker.

In a report in this month's Biochemistry, the scientists describe the key snafu in cystic fibrosis transmembrane conductance regulator (CFTR), a protein that regulates cellular salt levels and helps ward off bacteria in the lungs. They also offer a new way to remedy it.

Much like, when folding a big bed sheet, one little slip can turn the whole thing into a misshapen mess, a similar thing happens in CF patients, deep within their cells. A genetic slip deletes a tiny but essential slice of CFTR.

This deletion -- of a single amino acid along a chain of nearly 1,500 of them -- occurs at a critical juncture in the twisting, turning protein. So instead of folding into an orderly shape, part of the molecule comes undone.

Molecular traffic cops, which act as chaperones, catch the misshapen proteins and keep them from passing onto the cell's surface, where they are to shuttle chloride ions and other essentials into and out of the cell. The broken protein gets flagged, degraded and recycled.

"The deleted amino acid is like a passport," says Young-Hee Ko, Ph.D., who initiated the project. "Without it, the protein can't travel to the cell membrane, where it is critical for killing bacteria, especially in the lungs." Subsequently, CF patients suffer a lifetime of chronic lung infections and an early death.

The key step in the work was isolating a small, manageable section of the huge CFTR molecule. Because it would have taken years to decode the structure of the whole protein in the lab, the researchers saved time by synthesizing a small section, just 26 amino acids long. They also made a 25-amino acid version that was missing the same amino acid CF patients lack. That way, they could bypass the unwieldy 1,500-amino acid structure and focus on the smaller fragments. It was like turning a 1,500-piece puzzle into a 25-piece puzzle.

Next, colleagues Michael Massiah, Ph.D., and Albert Mildvan, Ph.D., used a technique called nuclear magnetic resonance spectroscopy (NMR) to determine, or "see," the structure of the two protein fragments. The NMR studies (see graphic) clearly showed the mutant, 25 amino acid segment in poorly structured, random shapes, and the normal segment in what senior researcher Peter Pedersen, Ph.D., calls a "beautiful helix."

The normal CFTR fragment (top) curls into a helix, while the mutant fragment unravels into random shapes, blocking it from migrating to the cell surface where it would prevent symptoms.

That's when the researchers knew CF was a "folding disease" -- much like sickle cell anemia -- and struck on the idea of fixing the problem by correctly folding the mutant protein.

In work yet to be published, Ko used an unlikely molecule -- heavy water -- to fix the mutant CFTR fragment. The heavy water acts like an extra set of hands on the bedsheet and folds the defective fragment into the same helical shape as its normal counterparts. And while heavy water is unlikely to help patients because of its toxicity, researchers now have a simple "test-tube test" to screen possible new CF drugs.

"We've finally confirmed what we proposed eight years ago, that most cases of cystic fibrosis are a result of a protein-folding problem," says Pedersen. "And now that we know what is broken at the most basic, chemical level, we can lab test non-toxic drugs -- there are millions of them that have already been studied -- to see if they fold the protein. It's the first time this kind of approach has been available."

Francis Collins, director of the National Human Genome Research Institute, and his colleagues first identified the CFTR mutation in 1989 and subsequently cloned the responsible gene. A flurry of research followed, much of it focusing on gene therapy, which tries to correct the genetic defect in the nucleus of cells, upstream of the chain of events that make the CFTR protein. Several gene therapy clinical trials have been conducted, but Pedersen says the early over-emphasis on this approach reduced funding to study CFTR.

"One alternative to gene therapy is getting outside the cell and showing that you can correct the problem in a test tube. And that's exactly what we've done," says Pedersen. "Then you can go back and see what happens inside the cell."

Seventy percent of CF cases are caused by the CFTR mutation studied by the Hopkins team. (The rest are caused by assorted CFTR mutations that their work does not address.) It is a recessive trait; to have CF, children need to inherit a defective copy of the gene from each parent. The disease affects 30,000 Americans and is most common among whites, where an estimated 1 in 20 carry the mutation.

The research was supported by grants from the National Institutes of Health and the American Lung Association.

Relevant Web sites:

Full-text of the scientific article in Biochemistry-http://pubs.acs.org/journals/bichaw/index.html
Hopkins Department of Biological Chemistry - http://www.med.jhu.edu/biochem
Pedersen's Home Page - http://www.med.jhu.edu/bcmb/faculty/pedersen.html
Cystic Fibrosis Foundation - http://www.cff.org


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