Target For Cystic Fibrosis Drugs Found

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 inthe lungs. They also offer a new way to remedy it.

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

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

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

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

The key step in the work was isolating a small, manageable section of thehuge CFTR molecule. Because it would have taken years to decode thestructure of the whole protein in the lab, the researchers saved time bysynthesizing a small section, just 26 amino acids long. They also made a25-amino acid version that was missing the same amino acid CF patientslack. That way, they could bypass the unwieldy 1,500-amino acid structureand focus on the smaller fragments. It was like turning a 1,500-piecepuzzle into a 25-piece puzzle.

Next, colleagues Michael Massiah, Ph.D., and Albert Mildvan, Ph.D., used atechnique called nuclear magnetic resonance spectroscopy (NMR) todetermine, or "see," the structure of the two protein fragments. The NMRstudies clearly showed the mutant, 25 amino acid segment in poorlystructured, random shapes, and the normal segment in what senior researcherPeter Pedersen, Ph.D., calls a "beautiful helix."

That's when the researchers knew CF was a "folding disease" -- much likesickle cell anemia -- and struck on the idea of fixing the problem bycorrectly 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 ofhands on the bed sheet and folds the defective fragment into the samehelical shape as its normal counterparts. And while heavy water is unlikelyto 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 casesof cystic fibrosis are a result of a protein-folding problem," saysPedersen. "And now that we know what is broken at the most basic, chemicallevel, we can lab test non-toxic drugs -- there are millions of them thathave already been studied -- to see if they fold the protein. It's thefirst 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 andsubsequently cloned the responsible gene. A flurry of research followed,much of it focusing on gene therapy, which tries to correct the geneticdefect in the nucleus of cells, upstream of the chain of events that makethe CFTR protein. Several gene therapy clinical trials have been conducted,but Pedersen says the early overemphasis on this approach reduced fundingto study CFTR.

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

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

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

--JHMI--

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

A related graphic is available online: http://hopkins.med.jhu.edu/NewsMedia/press/1999/JUNE99/990623.HTM

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