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MICROBIOLOGY Combinatorial Genetics Enlisted in Search For New Antibiotic DrugsHarvard Scientists Develop Method Useful to Basic Science, Drug Discovery As drug-resistant microbes render useless one antibiotic after another, an emerging medical crisis is forcing researchers to confront anew a question that once appeared to be settled: where are those microbe-thwarting substances that surely must exist somewhere in nature or in the synthetic chemist's toolbox? Adding a new way to tackle this problem is a study by Jonathan Blum, HMS postdoctoral fellow, and John Mekalanos, the Adele Lehman professor and chair of microbiology and molecular genetics at HMS. With Simon Dove and Ann Hochschild, instructor and associate professor, respectively, in the department, the researchers present in the Feb. 25 Proceedings of the National Academy of Sciences a method that can identify inhibitors of essential processes in bacteria.
Aiding the search for antibiotics, Simon Dove, Ann Hochschild, John Mekalanos, and Jonathan Blum devised a way to introduce proteins into bacteria and pick out those that harm the bugs. Conceptually, the technique somewhat resembles combinatorial chemistry, a set of methods widely used in drug discovery that is based on first creating libraries of diverse chemical compounds and then picking out the ones that do something interesting. Rather than chemically synthesizing nonpeptide molecules, however, the new approach exploits nature's ease at creating vast sequence diversity in peptides. In essence, it offers a low-tech version of a high-throughput screen that can be conducted by just a microbiologist with his petri dishes, without the need for chemists or robots, says Blum. Dubbed ABBIS (aptamer-based bacterial inhibition systems), the method uses a Trojan horse stratagem. It sneaks into bacteria a DNA library for random peptides, which then are expressed and go on, in some cases, to kill the bacteria or halt their growth. This way, ABBIS manages to test in a relatively simple experiment the 10 million substances contained in its DNA library, whereas even a large commercial combi-chem library contains only about two million compounds. Moreover, testing the random peptides inside bacteria also circumvents the problem that the bacterial cell wall keeps out many substances thrown at it in conventional searches for antibiotic substances. "ABBIS has the potential to help us find new targets and inhibitors of those targets. It could become important for developing new kinds of antimicrobials," says Blum. An MD-PhD, Blum sees patients with AIDS and other infections at Massachusetts General Hospital in addition to his research with Mekalanos. Basic researchers can also use the system with great flexibility for discovering inhibitors of their cellular process of interest. Targets and BulletsCollectively, today's antibiotics act against a small number of targets in a handful of biological processes, including the synthesis of the cell wall, RNA, or proteins. Yet previous work by the Mekalanos lab (see Focus June 4, 1998) has estimated that some pathogens need roughly 20 percent of their gene products to survive. These are all potential targets, but current antibiotics affect only a tiny fraction of them.On this petri dish, Jon Blum performed a simple test to see if a compound fished out of a DNA library actually blocks bacterial growth. He dipped a circular piece of filter paper into a solution of the sugar arabinose, let it dry, and placed it (inner circle) in the middle of a dish coated with an invisibly thin layer of bacteria. The bacteria harbor DNA encoding the compound, and arabinose can switch on its expression. As far as the sugar diffused out (clear ring), bacteria were unable to grow into the dense lawn they form elsewhere (gray outer ring). Image provided by Jonathan Blum ABBIS falls into the less conventional of two major camps dividing the field of drug discovery, says Blum. The more popular view is empirical. Most pharmaceuticals used today fall into a surprisingly limited number of structural groups that can penetrate cells and have good pharmacological properties. Therefore, most scientists search for new drugs among the relatives of old ones. Researchers in the other camp, however, scour the diversity of all nature's compounds and hope to solve pharmacological problems after a promising new compound is found. ABBIS, says Blum, stakes out an extreme version of this second view. A description of his experiment makes this clear. First, Blum constructed a genetic vector for ferrying the diverse peptides into bacteria. He took one developed in the laboratory of Jon Beckwith, the American Cancer Society professor of microbiology and molecular genetics at HMS, and modified it so it could be transferred more efficiently into pathogenic microbes. He then added a carrier protein called thioredoxin, into which he cloned random DNA sequences encoding peptides of 16 amino acids each. These are made by synthesizing oligonucleotides without imposing a specific sequence, that is, either an adenine, thymine, guanine, or cytosine can attach at each position. At 20 amino acids raised to the 16th power, this generates a theoretical diversity that far surpasses anything the scientists could, in practice, have cloned and then screened, says Blum. That's why the scientists assume that no two of the ten million random peptides they cloned into the library are alike. The method for inserting random peptides into thioredoxin—creating constructs dubbed aptamers—was first developed for yeast by Roger Brent, then an HMS faculty member (see Focus, May 10, 1996), and is being used in different forms by scientists in academia and biotechnology companies. Blum and Mekalanos are the first to apply it to bacteria. Living LibraryThe Harvard scientists searched their library in two ways that represent common situations in drug discovery. In both cases, they inserted it into E. coli and turned on expression of the aptamers.First, they checked whether the library contained an inhibitor against a known target, the enzyme thymidylate synthase (TS). It served mainly as a model to test the method's worth, but also has potential medical value as a target for antibiotics and cancer therapeutics. Blum used a genetic selection, that is, an experiment that allowed the growth only of bacteria in which an aptamer inhibited TS. That search turned up one aptamer, and further tests with Dove and Hochschild showed it probably worked by interacting directly with the enzyme. Blum is now working to improve this aptamer's binding strength to TS, and hopes that other researchers will obtain a crystal structure of the two bound together as the next step in a drug discovery effort. Second, Blum screened the library for aptamers that inhibit the bacteria's growth or kill them (see image). He found five, and now faces the task of figuring out how they work. The authors acknowledge ABBIS's limits. Proteins make lousy drugs: they are digested when swallowed, don't enter cells well when injected, provoke immune reactions, and are costly. Converting a protein with a desired biological effect into a drug formulated as a little pill remains a formidable, though not impossible, task. This means ABBIS can hand researchers new targets and some structural hints on what an inhibitor for them should look like, but the road from there to a new antibiotic is still long, the scientists agree. —Gabrielle Strobel |
