Seeking superbug-busters

Superbugs that can overcome the last remaining antibiotics. Bioterrorist threats of anthrax spores engineered to fend off all antibiotics. We’re on the brink of a postantibiotic era and yet have seen only one new antibiotic class come out of the drug pipeline in 30 years.

Now for the bad news. Major players in the pharmaceutical industry have been steadily moving away from anti-infectives R&D since the 1980s and are not showing any sign of making a comeback.

In 1969, then-U.S. Surgeon General William Stewart declared before Congress that it was “time to close the book on infectious disease.” By the 1980s, that widely held sentiment was shared by the pharmaceutical industry, which started drastically cutting back its anti-infectives research programs aimed at developing new antibacterial and antifungal agents. With infectious diseases sufficiently conquered, the need to hunt for new antimicrobial classes was no longer necessary, they reasoned. They were wrong. Fortunately, there is a silver lining in the pipeline. Small biotech firms are taking up the search.

Drug development
Currently, most pharmaceutical companies are limiting their anti-infective programs to building on existing classes of antibiotics—developing analogues that lead to second and third generations of the original compound. But overcoming resistance comes with a price, says Skip Shimer, vice president of research at Cubist Pharmaceuticals (Lexington, MA, www.cubist.com). “Most anti-infective [development] has been driven by chemists, and the chemists thought, ‘We can improve them.’ But you get to a point where you can’t rescue those compounds anymore. As you try to tweak and alter compounds to overcome resistance, you start to lose the bacterial spectrum. That’s not the way you treat infectious diseases. Your compound has to get all organisms, or physicians won’t use them.”

Furthermore, cross-resistance to the newer agents can develop in short order because of an abundant pool of existing resistance genes. Some microbes that are resistant to one of the original antibiotics harbor resistance mechanisms that recognize the newer agent on the basis of its structural similarity to the parent compound.

With the majority of antimicrobial classes discovered during the 1940s and 1950s, the golden era of antibiotics as “magic bullets” continued unhampered through the 1970s, with nearly all infectious diseases responding to the available arsenal of antibiotics. But by the 1990s, the presence of superbugs—bacteria resistant to one or more antibiotics—became an endemic fixture.

Penicillin

“A lot of the companies made their hay [from antibiotic discovery] during the 50s and 60s,” says Peter Hecht, CEO of Microbia (Cambridge, MA, www.microbia.com), a privately held biotechnology company. “Then everyone declared victory in the war on microbes, so over the last 20 years they’ve been getting out of anti-infectives. People thought they’d solved the problem.”

Only one new class of antibiotics has been added to the arsenal in 30 years. In April 2000, the FDA approved a new antibiotic called linezolid (Zyvox), the flagship drug of the new oxazolidinone class of antibiotics. As the first synthetic antibiotic class, scientists reckoned that the development of resistance to this fresh-faced addition would be unlikely. The existing antibiotic classes originated from soil microbes, which churn them out in nature to mark their territory amid microbial competitors. But microbes had never encountered the oxazolidinones before, clinically or in nature. One year later, disturbing reports surfaced of treatment failure stemming from staphylococci and enterococci that had mutated and become resistant to this last-resort antibiotic.

Methicillin

The bottom line
Yet the threat of crafty microbes developing resistance to drugs fresh out of the pipeline is not the reason Big Pharma has been pulling out, says Hecht. It’s return-on-investment, he explains. The continual merging of big players in the pharmaceutical industry means that pharmaceutical goliaths need billion-dollar blockbusters to achieve the requisite return-on-investment, rendering a $600 million drug useless to them. “Pharmaceutical companies want a 20% return-on-investment. But with companies that big, they need to generate enormous profits,” says Hecht.

“It’s striking to see how big pharmaceutical companies are disinterested,” agrees Mario Thomas, chair and CEO of Entomed (Strasbourg, France, www.entomed.com), an anti-infectives biotech company. “The reason is the size of the opportunity.”

Despite a worldwide market of $25 billion netted from antimicrobial sales annually, many antibiotics can generate an impressive $300 million but will not hit the billion-dollar mark. Even so, that is not to say the potential isn’t there. Some antibiotics continue to be billion-dollar babies for Big Pharma companies such as Hoffman-LaRoche, maker of the cephalosporin antibiotic Rocephin.

Vancomycin

Although we are disturbingly close to the brink of a postantibiotic era, this worrisome trend is not showing signs of reversing any time soon. Complicating the financial equation is the need to restrict the use of powerful new antibiotics to preserve their efficacy against superbugs when all else fails. Where vancomycin was once the antibiotic of last resort, the endemic spread of vancomycin-resistant enterococci in U.S. hospitals has created a scenario in which some strains are no longer treatable. Now, linezolid and the semisynthetic streptogramin antibiotic quinupristin-dalfopristin (Synercid), manufactured by Aventis, have filled the ranks as last-resort antibiotics against vancomycin-resistant superbugs.

For newly developed antibiotics, this can mean profit potential being wiped out from the get-go, which does not give Big Pharma any incentive to relaunch costly R&D programs. “I don’t see this changing from Big Pharma. . . . They’re not going to address a fragmented market,” says Thomas. “The opportunities do not meet with the financial targets of the big companies.”

Driven by the need for a huge return-on-investment, drug companies are instead focusing on drug discovery for chronic conditions such as autoimmune disorders and cardiovascular disease. The cholesterol-lowering statins, for example, garner $6 billion a year. “Those are drugs for 35- to 40-year-olds who will take them once a day for the rest of their lives,” points out Hecht.

But there is a silver lining. Thomas explains that this trend spells good news for the biotechnology industry. “It’s a great opportunity for small companies like us [Entomed], because with large companies abandoning research and development in anti-infectives, it has spawned a new group of young start-up companies to tackle the problem.”

Beetle-mania
For Thomas, that means tapping into the immune system of insects for potential new anti-infectives. To obtain these molecules, Entomed studies insects in countries near the equator. Although the immune systems of the more than 2 million insect species generally function the same way, each species has evolved its own library of molecules that Thomas says has provided Entomed’s researchers with vast new sources of potential compounds. They have never found the same molecule twice from one species to the next.

To trigger their immune systems, insects are challenged with a variety of microbes, which results in peptides being released in the hemolymph, the equivalent of blood in insects. The various peptides are then separated by high-pressure liquid chromatography and screened against seven different microbes. From there, researchers select the best hits, identify their chemical structures, and perform in vivo modeling.

“I spent 14 years in Big Pharma, and we still shake our heads why anyone hasn’t thought of that before,” marvels Thomas. “Nobody had deduced tapping into the immune response in the late 1980s, at the time when Big Pharma were abandoning research from natural sources for combinatorial chemistry. Why [would Big Pharma] start running after flying insects?” he quips.

So far, Entomed’s scientists have identified hundreds of compounds with antimicrobial properties. After selecting the best ones, they modify them through molecular evolution or chemical lead optimization to improve their profile against human pathogens. Thomas says that they now have a robust platform in which 25% of the compounds they have discovered are anti-infectives. The rest are biologically active molecules, some of which exhibit antiproliferative activity against cancer cells or show anti-inflammatory activity.

Half of the drugs on the market manufactured by pharmaceutical companies are derived from plants and microbes, says Thomas, which comprise just 23% of species contributing to biodiversity on the earth. Insects, on the other hands, make up 67% of the earth’s biodiversity. “Logic tells us that there’s got to be a few blockbusters out there,” he reasons.

Biofilm bonanza
Microbia has similarly carved out a niche in anti-infectives R&D by focusing on the problem of microbial biofilm formation, a phenomenon that can render the most potent antimicrobials completely useless. Biofilms are elaborate networks of bacterial cells that communicate with each other once their numbers reach a certain threshold. They can form on any attachable surface, such as in-dwelling medical devices and industrial equipment, the lungs of cystic fibrosis patients with pneumonia, and other organs. Film formation enables bacteria to survive stressors such as antibiotic therapy and to become up to 1000 times as resistant to antibiotics as free-dwelling cells. The Centers for Disease Control and Prevention (Atlanta, GA) estimates that biofilms are involved in 65% of infections.

Hecht explains that Microbia’s goal is to elucidate crucial targets in the genetic machinery that enable microbes to form these impenetrable films and develop compounds that cripple those targets. The company envisions developing bacterial biofilm inhibitors that would be administered alongside antibiotic therapy with the aim of resensitizing intrinsically resistant bacteria in the biofilm to the antibiotic being used. Microbia is also developing novel antifungal agents by identifying important genetic targets responsible for fungal virulence.

Like Thomas, Hecht sees the reluctance of Big Pharma to continue in anti-infectives as potentially good for biotechnology. Nonetheless, he wonders whether this trend could harm smaller companies. “It remains to be seen whether partnering opportunities go away,” he explains.

Strain bedfellows
Partnering opportunities are one way that some Big Pharma companies are keeping their hands in the pot. Some companies are even spinning off their anti-infectives departments into biotechnology companies under a different name. Such a move provides a new vehicle for raising money, points out Shimer.

Cubist is collaborating with pharma giant Novartis and is focusing on developing anti-infective compounds that show no cross-reactivity with known antibiotic resistance mechanisms. This collaboration centers around Cubist’s VITA technology, which identifies peptides that inhibit microbial targets and then validates whether the peptide inhibits growth of the microbe or inhibits the functioning of that target through expression of the peptides in the bacteria, essentially validating both peptide and target early in the drug discovery process.

Shimer says that Cubist is using a more rapid approach to drug discovery than that traditionally used by big drug companies. It has collaborated with Syrrx (www.syrrx.com), a San Diego-based biotechnology company specializing in high-throughput structure determination.

“Traditionally, you had to do a lot of work up front to identify a good series of compounds, then start to query more about the spectrum—how many [bacterial] orthologues would they inhibit,” explains Shimer. “You had to have an advanced program before beginning crystallization trials and had to do all that chemistry work in the dark.”

In Cubist’s approach, antimicrobial hits are identified by high-throughput screening and then co-crystallized with the same target from many different bacteria. This approach is crucial, explains Shimer, who says that although a target may serve the same function in different microbes, subtle differences determine a compound’s spectrum of activity. “We’re getting all the structural information early in the program.”

Cubist’s current lead compound, daptomycin, is the first product in a new class of antibiotics called lipopeptides, which consist of 13-amino-acid cyclized peptides and a hydrocarbon lipophilic tail. Daptomycin works by burying itself in the bacterium’s outer membrane and triggering a rapid depolarization and energy loss, causing potassium ions to spill out of the bug. Cubist expects to file an NDA (New Drug Application) with the FDA for daptomycin and hopes to see the product approved for market by 2004.

But while biotechnology companies lack the deep pockets of the big players, the advantage they have over the pharmaceutical giants is their focus, says Shimer. Cubist is intent on keeping ahead of the inevitable by focusing efforts on laboratory-generated mutants of bacteria that are resistant to daptomycin. Shimer’s team uses these strains to flesh out the part of daptomycin responsible for its activity to develop new compounds with better activity.

Their timing could not be better. We are running out of options faster than new compounds can hit the clinical setting. “We just get reminded that we share the world with microbes,” says Hecht. “Infection is not going away.”

Holding back the tide
To preserve the efficacy of antimicrobial agents, forward thinking is critical, says Fernando Baquero, a microbiologist at Ramon y Cajal Hospital in the National Institutes of Health (Madrid, Spain). Speaking to scientists at the 41st Interscience Conference on Antimicrobial Agents and Chemotherapy, held in Chicago last December, Baquero explained that the failure of drug companies to look beyond the end of the pipeline threatens to reduce the lifespan of antimicrobial classes. He says that drug companies continue to make this mistake. “Companies are not very interested in promoting knowledge of how resistance occurs,” he explains. “That's the problem. There has been nothing except simple experiments [to detect resistant mutants], and these are totally unreliable.”

Traditionally, screening for resistant mutants has entailed challenging bacteria with increasing concentrations of an antibiotic and selecting those that have evolved tolerance. That is not enough, says Baquero. “We should determine all targets of an antibiotic and be able to detect all possible mechanisms of resistance, and determine if antibiotics will trigger genetic variation.” Detecting such variations, he explains, can tell scientists the extent of a resistance threat. The mutation may initially be costly yet ultimately temporary, disappearing from a population within successive generations.

The use of microarrays is one approach that can determine how bacteria will respond to an antibiotic, he says, by enabling detection of preresistance genes. These are genes that are not entirely fit for resistance but can evolve into full-fledged resistance genes under selective pressure. Similarly, the arrays can determine if efflux pumps are turned on in the presence of the antibiotic or if inducible genes are hyperexpressed.

Computer modeling of the target molecule and antibiotic can be used to determine what modifications will prevent the antibiotic from interacting with the target and can be followed up with in vivo testing. Hypermutable strains are another approach that can jump-start nature and provide an earlier glimpse into nature’s bag of tricks. Hypermutable bacteria are 1000 times as adaptable to external changes as normal strains. “These bacteria are extremely useful to predict what will happen within days what in nature might take years. Once you have an adapted bacterium, you can see how it has adapted,” says Baquero.

And finally, known resistance genes in nature can be mobilized into laboratory bacteria to determine if existing mechanisms protect the bugs against the new compound. “An antibiotic may be new for us but not be new in nature,” he points out. “Resistance genes may already occur in nature, . . . [but] we can predict what will happen in the real world.” With the technologies now available to make these predictions, Baquero warns that the most promising compounds may be compromised soon out of the pipeline if anti-infective makers do not take precautions to outwit microbes at their own game of survival.

Further reading

Cassell, G. H.; Mekalanos, J. J. Am. Med. Assoc. 2001, 285, 601–605.

Guillemot, D.; et al. Clin. Inf. Dis. 2001, 33, 542–547.

Walsh, C. Nature 2000, 406, 775–781.

Nicole Johnston is a freelance writer and biochemist at McMaster University (Hamilton, ON). Send your comments or questions regarding this article to mdd@acs.org or the Editorial Office by fax at 202-776-8166 or by post at 1155 16th Street, NW; Washington, DC 20036.