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MOLECULAR PHARMACOLOGY Enzyme Pair Joins Fight Against Drug-resistant Bacteria Researchers Create New Version of Vancomycin HMS researchers, working with scientists at other institutions, have overcome a preliminary yet critical hurdle in the push to develop antibiotics against drug-resistant bacterial strains. Most attempts have been plagued by a lack of molecular tools for manipulating--and ultimately improving--the structure of naturally occurring antibiotics. Heather Losey, Christopher T. Walsh, and their colleagues report in the Dec. 16 Chemistry & Biology that they have harnessed two enzymes, which work by adding sugars to a central molecular core, and used them to create new versions of two potent antibiotics, vancomycin and teicoplanin. After using a pair of enzymes to modify the structure of potent antibiotics in a test tube, a future step for Heather Losey and Christopher T. Walsh may be to see if they can manipulate bacteria to produce the redesigned drugs. (Photo by Graham Ramsay) It is not clear if the new versions are effective against drug-resistant bacterial strains. "These drugs are not going to go on the market any time soon, but that was not our goal," said Losey, HMS graduate student in biological chemistry and molecular pharmacology. "This is just the first step out of many to find out if we can tweak the molecules." What surprised Losey and Walsh, the Hamilton Kuhn professor of biological chemistry and molecular pharmacology at HMS, and their colleagues was how much tweaking they could do with the enzymes. One of them, glycosyltransferase E, was capable of transferring a wide variety of sugars to the central core of the antibiotic molecules. "This just gives us more tools, more permutations to try," said Losey. Though still years away, getting bacteria to manufacture drugs that humans have designed is not in the realm of science fiction. And flexibility will be needed in the fight against drug-resistant bugs, especially those unfazed by vancomycin. An antimicrobial powerhouse, vancomycin was used for nearly half a century as a last line of defense to fight bacteria that were resistant to other antibiotics. Overuse, especially in hospitals to control staph and enterococcal infections, has allowed hardier pathogens to flourish. As new vancomycin-resistant strains emerge, researchers will have to devise new and smarter drugs to fight them. Sugar Solutions Though the results of their enzyme-wielding experiments have not been tested against drug-resistant strains, Losey and her colleagues believe that altering sugars may produce more effective antibiotics. To begin, certain sugars can increase the solubility of molecules. They can also provide sites for the attachment of other molecules that in turn might enhance a drug's antimicrobial properties. One antibiotic in phase III clinical trials, oritavancin, appears to use this principle in fighting vancomycin-resistant strains. It is essentially vancomycin with a bulky molecule appended to a sugar. The pharmaceutical giant Eli Lilly created the molecule by randomly adding chemical groups to the vancomycin core. Losey and colleagues wanted to see if they could intentionally design a similar molecule and possibly improve upon it. Like many antibiotics, vancomycin works by interfering with a pathogen's ability to make a new cell wall. To do so, it must first bind to a short stretch of amino acids. Some strains have the ability to change the structure of the amino acid stretch in the presence of vancomycin, preventing the antibacterial agent from gaining a foothold. Thinking that oritavancin might bypass this chain of events by bringing vancomycin directly to the nascent cell wall before the bacterial genes could be turned on, Losey and colleagues set out to create a similar molecule. It required first adding a sugar to the vancomycin scaffold, traditionally not an easy task. "It is really hard to put these sugars on chemically," said Losey. To carry out their plan, the researchers decided to harness the same compounds that vancomycin-producing bacteria use to add sugars, namely the glycosyltransferases. Having isolated the genes for several of these enzymes, including GtfE and GtfD, the researchers purified the enzymes. But they faced a challenge. Oritavancin's ability to defeat drug-resistant strains appears to be a function of the bulky chemical group appended to the sugar, rather than the sugar itself. The real stumbling block was finding a sugar that could be added to the vancomycin core at the right place and with the right chemical structure for accepting a chemical appendage. Versatile Enzyme Tools As it turns out, Daniel Kahne of Princeton and Jon Thorson of the University of Wisconsin had each developed multiple forms of two main classes of sugar-donating substrates. The HMS researchers exposed each of these glucose derivatives to GtfE, which was known to transfer sugars to a critical spot on the vancomycin scaffold. The pairings were remarkably successful. "We found we could use GtfE to transfer any of these glucose derivatives to the vancomycin and teicoplanin scaffolds," said Losey. Using a second enzyme, GtfD, they were able to add additional sugars, creating disaccharide bonds that in turn provide sites for additional chemical groups. "This shows the potential for in vitro synthesis using enzymes, not chemicals, to put derivatives of sugars on a teicoplanin and vancomycin scaffold," said Losey. Having taken the first steps toward creating oritavancin-like molecules in a test tube, Losey foresees the day when they may be made in bacteria. "If we can have the organisms somehow make these structures that we have shown are better, we should have an easy way of producing new antibiotics," she said. Though still years away, getting bacteria to manufacture drugs that humans have designed is not in the realm of science fiction. "The glycosyltransferases are normally found in vancomycin-producing organisms," Losey said. "We would have to put in a set of genes that are responsible for making alternate sugar substrates. The hardest part will be to figure out how to make those nucleotide sugars in organisms." --Misia Landau