By Sean Henahan, Access Excellence

SANTA CRUZ, CA (NOVEMBER 21, 1996) A new atomic view of the interactions between common antibiotics and bacteria they target, should help researchers to better understand antibiotic resistance and to design more effective drugs.

UC Santa Cruz biochemist Joseph Puglisi and colleagues worked for more than two years to determine how antibiotics grab a bacterium's ribosomes--the factories in every cell that make the proteins an organism needs to survive. The team used a technique called nuclear magnetic resonance (NMR) spectroscopy. NMR probes the relative positions of atoms held in a magnetic field by tickling particular atoms with pulses of radio waves. A computer analyzes the data to predict the most likely positions of atoms with respect to each other.

Using NMR the scientists were able to map how the ribosome interacts with the antibiotic paromomycin, one of a class of naturally occurring antibiotics called aminoglycosides.

"Puglisi can see precisely how the antibiotic binds to the ribosome at the atomic level," says Harry Noller, director of UCSC's Center for the Molecular Biology of RNA and a member of the National Academy of Sciences. "This explains for the first time how a ribosome-directed antibiotic works. Since these are among the most widely used antimicrobial drugs, Puglisi's result is of major medical and scientific significance."

The researchers solved the structure of the drug attached to a short bit of RNA--a single-stranded relative of DNA--from the most critical part of the bacterial ribosome. Other scientists had probed how antibiotics link to proteins, but none had deciphered an antibiotic-RNA complex.

"There's a big rebirth in the idea of targeting RNA in cells by using small molecules," Puglisi says. "This is an example of how these 'lock-and-key' systems work. Manipulating the details of this system suggests a strategy for a whole new field of RNA-drug interactions."

The final solution is the "best fit" of twenty structures churned out by the computer as good matches to the data. In three dimensions, the structure reveals that the ribosome forms a small pocket into which the L-shaped antibiotic molecule fits precisely. Chemical groups at several spots interact to "glue" the two units together.

This level of scrutiny allowed Puglisi and his coworkers to address several issues:

How the antibiotic gums up protein manufacture and--by extension--how the normal ribosome functions. The tiny paromomycin molecule zeroes in on the "decoding" region, where the ribosome interprets genetic messages from the cell's nucleus and strings together the building blocks of proteins in the correct order. After it fastens, the antibiotic makes the ribosome hold onto each building block too long. The ribosome then makes errors as it attempts to read the genetic code, leading to inviable proteins.

"We think there's a 'reading head' in the ribosome. The aminoglycoside comes in and makes it get sticky and sloppy," Puglisi says.

Why the antibiotic acts selectively on bacteria. The RNA studied by Puglisi's team consists of a 27-piece string of RNA's four basic units, which biologists identify with the letters A, C, G, and U. At one crucial spot, all bacterial ribosomes have an A, whereas all higher organisms--including humans--have a G.

This tiny evolutionary switch is enough to disrupt the pocket into which the antibiotic molecule clicks, so the fit isn't as tight in people as it is in bacteria. Still, the antibiotic does work in people at low levels, leading to occasional side effects such as deafness and kidney damage.

How some antibiotic resistance arises. A single change in either the RNA sequence of the bacterial ribosome or the structure of the antibiotic molecule can prevent them from fitting snugly.

The usual scenario, Puglisi's says, is for the bacteria to attack the antibiotic with an enzyme that alters one of the most vital chemical "glue" groups. Once that mechanism evolves, it spreads readily among the promiscuous bacteria via genetic transfers.

"Our research shows exactly which parts of the structure are important to the drug's function," Puglisi says. "So, we can try to vary the other parts to come up with versions that are less toxic to humans and less prone to resistance." Puglisi believes the pharmaceutical industry will see these potential benefits. "This class of antibiotics was revolutionary when they were discovered, but they are no longer the drugs of first choice," he says. "Now we have a logical framework to try to make them more useful again.

Aminoglycosides including gentamicin, kanamycin, tobramycin, and neomycin fhave become less effective in treating infection as antibiotic resistance has spread. The new finding should help researchers understand how bacteria manage an end run around the drugs by developing resistance to them.

The research appears in the November 22, 1996 issue of the journal Science