By Dean A. Haycock
Special to BioWorld Today
The appropriateness of the phrase ¿the good old days¿ depends on which aspect of those days you have in mind. Times may have been less hectic before fax machines, cellular phones and e-mail, for example, but they were hardly trouble free. It was not unusual before antibiotics became available to die from what is today considered a simple infection. The emerging phenomenon of antibiotic resistance, however, is threatening to bring those ¿good old days¿ back unless new antibiotics can be identified.
¿An increase in the size of the arsenal of different categories of drugs, and different physiological pathways in the cell that they target, gives us many more options for the future and gives us a chance in this evolutionary race,¿ John Mekalanos, professor and chairman of the Department of Microbiology and Molecular Genetics at Harvard Medical School, told BioWorld Today. ¿It is a race that I think the bacteria have the upper hand in because of their division time.¿
This real bacterial advantage helped motivate Mekalanos and his colleagues to look for new ways to identify potential targets for new antibiotics. They report their progress in the current issue of the Proceedings of the National Academy of Sciences, dated Feb. 29.
In an article titled ¿Isolation of peptide aptamers that inhibit intracellular processes,¿ they describe the adaptation of a method ¿ originally developed in yeast ¿ to identify novel antibacterial drug targets. The technique involves the isolation of random peptides that inhibit essential intracellular bacterial processes. This is accomplished by expressing a collection of random peptides directly inside the bacterium being probed for weaknesses. The peptides expressed are fused with a bacterial enzyme, Escherichia coli thioredoxin. The peptide-enzyme pair is called an aptamer. The choice of thioredoxin as a partner for the peptides, and the term aptamer, can be traced to the pioneering work in yeast systems in which thioredoxin was used for aptamer construction.
¿The thought behind thioredoxin is that, first of all, it is a small protein,¿ Mekalanos explained. ¿It has 110 amino acids, approximately 10,000 molecular weight. That is about a third the size of the average protein. The thought is that stoichiometrically you can perhaps make more of that protein than you can other, larger proteins that might put more stress on a cell. So we suspected that it would be less toxic to overexpress the aptamers based on that protein than on other types of scaffold proteins.¿
Screening turned up five aptamers that affected bacterial growth. Four of these inhibited or retarded bacterial growth and one was lethal. Other manipulations indicated that these effects could most likely be attributed to the protein aptamers themselves and not to other compounds such as RNA in the cells.
Aptamer¿s Utility Seen in Target Discovery
¿We don¿t see the aptamers as drugs per se right now,¿ Mekalanos said, ¿although they could conceivably be designed into a drug if their active site is small enough and some kind of structural information is obtained about how the aptamer interacts with the target protein. But right now we see this as mainly a target discovery tool, a way of finding those targets that are most easily inhibited to produce the phenotypes that we are interested in, cell growth inhibition and death.¿
The approach has the potential to highlight previously unsuspected targets for drug developers since nature, Mekalanos pointed out, has tended to develop antibiotics that affect a relatively small number of biochemical targets in bacterial cells. These include the cell wall, folate metabolism, DNA gyrase, protein synthesis or RNA synthesis. Hence, the Harvard researchers point out in their paper, ¿The majority of essential genes are not targets of any known antibiotic. For Haemophilus influenzae, it has been estimated that about 20 percent of its genes are essential; clearly, most of these gene products are not affected by currently available antibiotics.¿
Few classes of antibacterial drugs can easily enter bacterial cells and stay there long enough to act, they added. The aptamer approach gets around this problem by expressing compounds directly inside bacteria.
¿When you find aptamers that kill lots of different organisms, that might say something about the broadness of that target. Or it might say something about how trivial the mechanism of that aptamer is. Someone could say it works like a detergent or something dissolving the cell membrane,¿ Mekalanos warned. But early results indicate that specific targets may be identified successfully using the aptamer approach.
There Could Be Benefits In Specificity
¿This is unpublished, but we have been finding that some of the aptamers that kill one organism don¿t kill another organism,¿ Mekalanos said. ¿This is not true for 100 percent but it is true for some of the aptamers. That would suggest in our minds that some of these aptamers are very specific in terms of the targets they are picking out. And that is an advantage if you are making a designer drug that you want to affect a narrow spectrum of agents so that you don¿t wipe out the normal flora and end up with other complications that occur with broad-spectrum antibiotics.¿
He believes that aptamer selection has the advantage of making the target selection process very general. ¿It is saying, Just give us inhibition. Let¿s not worry about the issues of cell permeability and things that are problematic for developing an effective drug. Let¿s just go ahead and develop the screen and method of asking nothing more than the downstream result of inhibition or death. Let¿s see what kind of targets we come up with,¿ Mekalanos said. ¿If this exercise produces the same targets exactly that nature has selected, then that says that the natural process has picked the weakest links in the chain to go after. If aptamer selection indicates a completely new target, then it probably would argue that there is nothing preventing us from making antibiotics against any essential gene product as long as we develop an appropriate plan of attack.¿
The Harvard researchers are now conducting negotiations to license their method of identifying potential new antibacterial drug targets.