The Frog Solution
Scientists are hunting for the next generation of antibiotics in frog skin and pig gut, in silk moths and salamanders, in snakes, sharks, and honeybees.
AT MAGAININ PHARMACEUTICALS, in leafy Plymouth Meeting, Pennsylvania, amphibians abound. A plastic toad guards the entrance, startling strangers with a mournful mechanical croak. A beanbag frog sits atop the desk of Magainin’s founder, Michael Zasloff. And in a tank that Zasloff keeps in his lab swim a clutch of African clawed frogs, Xenopus laevis. The fun begins when he lifts one out. Grasping the squirming frog firmly in his hand, Zasloff sprinkles a dried form of the hormone adrenaline on its back and rubs it in. Within seconds, hundreds of tiny white spots dot the frog’s skin, then slowly merge into a creamy sheen. Zasloff calls the white film “a beautiful bandage” because it’s filled with bacteria-killing antibiotic peptides–small strings of amino acids, which are the building blocks of all proteins.
The discovery of one of these peptides, which Zasloff named magainin after the Hebrew word magain, or “shield,” inspired him to found the company of the same name 11 years ago. He wasn’t looking for unconventional antibiotics at the time. As a physician and molecular biologist researching gene expression at the National Institutes of Health, he was harvesting immature eggs from Xenopus ovaries. Although he operated on the frogs in nonsterile conditions and then returned them to microbe-infested water tanks, their wounds never became infected or even showed signs of inflammation. Zasloff concluded that something in the frogs’ skin was protecting them from bacterial attack. So he took a piece of skin, ground it up, and extracted its protective component, which turned out to be a peptide. Magainin, he found, is discharged onto the frog’s skin in response to adrenaline, which is released when pain receptors in the skin send the brain a message that an injury has occurred.
Since that early find, Zasloff has discovered peptide antibiotics in all manner of places: the airway and tongue of a cow, the gut of a pig, the stomach of the spiny dogfish shark. Other researchers are in on the hunt, too, and they have found peptide antibiotics in a witches’ brew of beasts: in silk moths, fruit flies, honeybees, and budworms; in salamanders, snakes, and horseshoe crabs; in the white blood cells of pigs and cows and humans; in our skin; and in fish, birds, and even plants. “They are now recognized in every species of life, from the smallest to the biggest, from things that fly or swim or just sit around on rocks to human beings–all defend themselves with antimicrobial peptides,” says microbiologist Bob Hancock of the University of British Columbia in Vancouver. “And they’re a very large proportion of what protects us daily from infection.”
Hancock and Zasloff are among a handful of pioneers who are turning these peptides into antibiotic drugs in hopes of someday providing an alternative or an adjunct to currently available antibiotics. The need for new antibiotics is becoming increasingly urgent as misuse and overuse of conventional antibiotics has bred resistance in many common disease-causing bacteria. And so the possibility of new antibiotics with novel modes of action that may be immune to resistance is exciting news. Some of them are even active against some disease-causing protozoans, fungi, and viruses such as Herpes simplex and HIV. Still, it must be stressed that although some of these new peptides are undergoing clinical trials, none have yet emerged into general use, and many problems in their manufacture remain.
WHAT MAKES THESE NEW peptide antibiotics different from penicillin and its kin is that they stem from animals’ own immune defenses rather than from other microbes. That means they have spent millions of years in an evolutionary battle with invasive bacteria and have emerged victorious time and again. Moreover, their mechanism of action is entirely different from that of most conventional antibiotics. Instead of disabling a vital bacterial enzyme, as penicillin does, antimicrobial peptides appear to punch holes in bacterial cell membranes, making them porous and leaky. To develop resistance to conventional antibiotics, bacteria need only remodel an enzyme or two, changing them so that they still perform their function but no longer bind to the antibiotic. It’s far more complicated for a bacterium to completely remodel its membrane in response to pore-forming peptides.
“To make bacteria resistant to peptides,” says Hancock, “you need to change the entire composition of the membrane,” which means altering dozens of interrelated proteins. “So it’s very difficult.” For these reasons, advocates of peptide antibiotics believe that bacteria are far less likely to become resistant to them.
Peptide antibiotics are the first particles to protect us from the constant onslaught of invasive bacteria that we breathe in or swallow, or which simply land on our skin. They line every surface of the body–eyes, skin, lungs, tongue, and intestinal tract. They are found in the kidneys and in macrophages and neutrophils, white blood cells that use them to destroy engulfed bacteria. Peptide antibiotics are the rapid response troops that kill bacteria within minutes of encounter–far faster than antibodies or T cells, which can take a week or more to recruit and which do not exist in more primitive animals like insects. And most eukaryotic organisms–that is, every organism but bacteria and viruses–are immune to attack by these peptides because of a quirk in the makeup of their cell membranes. Eukaryotic cell membranes, which consist of fats and cholesterol, carry only a very low electric charge. But bacterial cell membranes, which are made up of a mixture of fats and sugars, carry a much stronger, negative charge. Because peptide antibiotics are positively charged, they are able to bind quickly to bacteria.
That’s when the killing begins. The interior of a bacterium is even more negatively charged than its exterior, and the strong attraction pulls the peptides into the cell membrane. There they adopt a helical form and gather into barrel-like clusters, each peptide acting as one stave in the barrel, with a hole in the middle of the cluster. The cell’s cytoplasm can now leak out through the holes, causing the bacterium to collapse. Furthermore, the holes can form entry points for conventional antibiotics, thus enhancing their action.
Unfortunately, not many peptide antibiotics are on their way to the clinic, and even those currently undergoing testing would have only limited use. One reason for the slow pace is the cost of bringing a drug to market, which has been estimated to cost about $300 million, including clinical trials. Another major expense is producing peptide antimicrobials in the first place. Grinding up frogs to extract magainin is dearly not an option, which leaves chemical synthesis–stringing together the relevant amino acids–or some form of recombinant production using genetically engineered bacteria. But there are problems with both approaches.
“When we started, the only techniques out there for production of therapeutic peptides were archaic chemical techniques,” recalls Zasloff. “This would lead to a cost for the peptide of about $1,000 to $2,000 a gram.” Improvements in chemical synthesis have brought the cost of magainin down to $100 a gram, but that’s still pricey. On the other hand, splicing a gene for the peptide into a bacterium that will be killed by its product doesn’t make sense either. Magainin Pharmaceuticals says it has bypassed the problem by fusing its peptide to a protein that renders it unable to attack bacteria until the protein is removed.
Despite the difficulties, some peptides are beginning the arduous trek to market. One of the most promising is a compound called BPI (for “bactericidal/permeability-increasing” protein), originally derived from human neutrophils and developed for clinical use by a small Berkeley, California, company called Xoma. In November 1997, Xoma published the results of a clinical trial of BPI for the treatment of meningococcemia, a virulent infection that affects nearly 3,000 children a year in the United States. This condition can progress from flulike symptoms to death within one or two days. The trial showed that BPI could significantly reduce the death rate from the disease, which is caused by a bacterium called Neisseria meningitidis. This bug releases a poison called endotoxin into the bloodstream. Endotoxin triggers a massive reaction, called a sepsis cascade, that in turn can cause severe inflammation and collapse of the circulatory system. The beauty of BPI is that it not only kills bacteria but binds to endotoxin, stopping the sepsis cascade. The endotoxin is then broken down by the liver and excreted.
ANOTHER PEPTIDE ANTIBIOTIC that’s winding through clinical trials–and should be available for use, if approved, in the fall of 1999–is a magainin derivative called pexiganan, an antibiotic cream for the treatment of infected diabetic foot ulcers. Untreated diabetic foot ulcers can lead to bone infections and ultimately amputation. Tests showed pexiganan to be effective in curing infection, and without the side effects–diarrhea and insomnia–that are common to the currently favored oral antibiotic.
There are other peptide antibiotics at similarly early phases, all being developed by small companies funded by venture capital. For example, the Vancouver-based Micrologix Biotech (a company with which Hancock is affiliated) produces peptides called bactolysins, originally derived from insects, which it claims can kill resistant strains of Staphylococcus aureus–in mice, anyway. IntraBiotics Pharmaceuticals, a company in Mountain View, California, is running human safety trials of peptides called protegrins, derived from ones originally discovered in the white blood cells of pigs, for the treatment of oral mucositis, a painful mouth infection often brought on by cancer chemotherapy. And Robert Lehrer, a UCLA physician also affiliated with IntraBiotics, is developing a protegrin-containing vaginal microbicide that could be used by women to prevent sexually transmitted diseases.
Obstacles remain. One is that peptide antibiotics given orally would be digested by intestinal enzymes, which are designed to pounce on proteins. That means that the antibiotics’ use would have to be restricted to injection or topical application. But even this hurdle can be overcome, says peptide chemist Bruce Merrifield of Rockefeller University in New York. A few years ago he synthesized a “mirror image” version of a peptide called cecropin, which is derived from the silk moth Hyalophora cecropia. The mirror-image peptide uses amino acids that are backward versions of those found in nature, which make it invulnerable to intestinal enzyme attack. But it can still kill bacteria, says Merrifield, including some in the same family as the bacteria that cause tuberculosis.
Another sticky issue is toxicity. “The antibiotic peptides are charged, which causes them to exhibit certain toxicities as the dose is increased,” cautions Zasloff. “I think they begin to irritate parts of the nervous system”–whose cells are also electrically charged–“so the therapeutic window is not as broad. That can be dealt with by chemical manipulation, and we are dealing with it.”
THEN THERE’S THE TOUCHY TOPIC of resistance. Most peptide researchers say that the potential for disease-causing bacteria to develop resistance to the peptides is small–and that try as they might, they can’t induce resistance in their target bacteria. Though Hancock concedes that a few species of bacteria are naturally resistant because their cell membranes lack a strong negative charge, they are not, he insists, of major clinical significance. It’s a view not shared by microbiologist Eduardo Groisman of the Howard Hughes Medical Institute and Washington University School of Medicine in St. Louis, who works on Salmonella typhimurium. “Salmonella has been isolated from a hundred different animal species,” says Groisman. “You name an animal–elephants, yes; camels, yes; cockroaches, yes. Who was isolating it from cockroaches or lizards, don’t ask me, but people have reported in reputable journals that Salmonella is there. Now, many animals that it infects have been shown to produce antimicrobial peptides against invading pathogens. So our rationale was this: since Salmonella has been isolated from many different animal species, it must have evolved ways to deal with these peptides.”
In fact, Groisman has found one strain of Salmonella–originally isolated from a cow it had killed–that can comfortably survive a dose of magainin. He claims that most peptide researchers who can’t find resistance aren’t culturing their bacteria under the right conditions and adds that Salmonella only expresses resistance when it needs it–that is, when conditions are similar to those it experiences inside the cells it infects. Those cells are macrophages, or scavenger cells, and the small, saclike organs in which they envelop Salmonella seem to be low in magnesium. Grow Salmonella in magnesium-deficient culture and they will survive peptide attack, says Groisman. Although he doesn’t know how Salmonella becomes resistant, he suspects that it may reduce its negative charge by modifying its membrane. Alternatively, it may secrete an enzyme that can chop magainin in two before the peptide can attack.
Despite his disturbing results, Groisman doesn’t believe that antimicrobial peptides should be dismissed lightly. For one thing, manufacturing them in mirror image, though expensive, may render them immune to resistance. And the supply of effective antibiotics is rapidly running out. “We really need new classes of antibiotics,” says Groisman, “and I think we should give them a chance.”
But could they ever replace the antibiotics to which so many bacteria have become resistant? Zasloff thinks they can as a last resort; however, he thinks it equally likely that they’ll be used to complement existing antibiotics, since the damage they do to bacterial membranes can open the door to other drugs. Clinical microbiologist Fred Tenover of the Centers for Disease Control and Prevention in Atlanta, who specializes in the molecular biology of antimicrobial resistance, paints a darker picture. “Every year there’s a meeting called ICAAC–the Interscience Conference on Antimicrobial Agents and Chemotherapy–and we see poster after poster of new drugs, some totally synthetic, some natural products, that inhibit wide ranges of bacteria at very low concentrations,” he says. “But three years later they’re never heard of again because they’re either too toxic or cause significant side effects, or trigger an immune response. So it’s hard to look into the crystal ball and say these really are the future of antibiotic therapy until we see how they do in well-controlled clinical trials.”
Nonetheless, Tenover remains hopeful. “We’re always looking for new ways to inhibit emerging infectious diseases,” he says. “Of course, one of our favorite ways is to prevent them in the first place with vaccines. But given that you can’t have a vaccine to everything, what do you do with the bacteria that remain that cause infections? And as many new things as we could have to throw at the bacteria are going to be helpful, because it’s our feeling that use eventually leads to resistance. Bacteria have been around for more than 3 billion years, and they’ve developed pretty good ways of figuring out how to overcome just about anything. So we’re happy to see lots of new things coming down the road.”
JOSIE GLAUSIUSZ (“The Frog Solution,” page 88) is an associate editor of DISCOVER. “This story has convinced me that the witches in Macbeth were onto something after all,” she says.
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