Infectious disease is back with a vengeance.Twenty years after the surgeon general of theUnited States claimed “We can close the bookon infectious diseases,” medicine appears tobe heading back to the preantibiotic era.According to the World Health Organization(WHO; Geneva), more than 95% ofStaphylococcus aureus strains worldwide arenow resistant to penicillin, and up to 60% areresistant to its derivative, methicillin.Resistance is spreading not only in hospital-acquired infections, but also in community-acquired pathogens such as pneumococciand tuberculosis. Alarmingly, in Japan,Europe, and the United States, there havebeen at least four cases of S. aureus resistantto vancomycin—the antibiotic of last resortin treating life-threatening infections.

This growing public health problem hasspurred renewed efforts to discover noveltypes of antibacterial agents with mecha-nisms radically different from existing com-pounds. With pharmaceutical companiesbanking on bacterial genomics to delivernovel targets for future antibiotics, severalsmaller companies are testing completelynew classes of antibacterial agents that targetbacterial cell membranes, adherence mecha-nisms, or gene expression. Although the clin-ical efficacy of many of these compoundsremains unproven, they offer some promiseof slowing the emergence of resistant strains.

End of an eraFew would dispute the profound impact ofantibiotics on human health. Indeed, muchof the eight-year increase in average humanlife span between 1944 and 1972 has beenattributed to their global introduction intomedical practice1. What is now consideredthe “antibiotic era” was ushered in when theindustrial-scale fermentation of penicillin in1943 enabled wide use of the antibiotic in theclinic. In the following two decades, the sys-tematic screening of natural productlibraries from soil samples or marine envi-ronments identified most of the classes ofantibacterial agents in use today.

When these approaches began to yielddiminishing returns in the 1960s, companiesturned instead to semisynthetic modificationof existing antibiotics to produce second-and third-generation compounds withbroadened antimicrobial activity, enhancedoral bioavailability, and improved toxicologi-cal and pharmacokinetic properties.

Today, antibiotics are the third largest-selling class of drugs, with a worldwide annu-al market estimated between $7 and $22 bil-lion. More than 150 antibacterial drugs havenow been approved in the United States, withmore than 27 new compounds currently inclinical development2.

Although these numbers are impressive,only one new antibiotic was approved by theUS Food and Drug Administration (FDA;Rockville, MD) in 1993, none in 1994, andonly a handful since3. Of particular concern,only two agents currently in the clinic—theoxazolidinones (which inhibit bacterial pro-tein synthesis initiation by binding the 50Sribosomal subunit) and cationic peptides(which permeabilize bacterial membranes)—act on unconventional targets. All the othersare merely analogs of earlier antibiotic classes,targeting a paltry 15 different bacterial targets3.

In fact, most current antibiotics arederived from a mere 15 or so base com-pounds, the main ones being the β-lactams(e.g., penicillins, methicillin, andcephalosporin), aminoglycosides (e.g., strep-tomycin, gentamycin, and neomycin),

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quinolones (e.g., ciprofloxacin), macrolides(e.g., erythromycin), lincosamides (e.g. clin-damycin), sulfonamides (e.g., sulfadiazine),tetracyclines (e.g., glycycline), and glycopep-tides (e.g., vancomycin). In the clinic, theseare categorized as broad- or narrow-spec-trum agents depending on their selectivityagainst Gram-negative and Gram-positivebacteria. Those compounds that are bacteri-cidal (lethal) tend to be preferred to bacterio-static (growth-inhibiting) antibiotics becausethe emergence of resistance is less likely.

It is now clear that reduced bacterial sus-ceptibility to second- and third-generationanalogs of existing structural classes has arisenbecause bacterial resistance mechanisms areso widely entrenched. As Stuart Levy, profes-sor of medicine, molecular biology, andmicrobiology at Tufts University School ofMedicine (Boston, MA) puts it: “The determi-nants for resistance are already in existence,it’s just a matter of time before they appear.”This, together with the pervasive and injudi-cious use of these agents (see “The rise of bac-terial resistance”) has exacerbated the devel-opment of drug resistance and hastened the

FEATURE

The new antibioticsCan novel antibacterial treatments combat the rising tide of drug-resistant infections?

Holger Breithaupt

Figure 1. Resistance is futile. A highly stylized cartoon of a bacterium showing the targets ofmechanisms of action of novel antibiotic treatments currently under development.

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obsolescence of newly introduced antibiotics.Public health authorities have responded

by establishing surveillance efforts to betterunderstand the epidemiology and emergingpatterns of resistance. Monitoring programshave been set up in the US by the Centers forDisease Control (CDC; Atlanta, GA) andglobally by the WHO to collate drug resis-tance data. In addition, the SENTRY antimi-crobial surveillance project, launched in1997 with funding from Bristol MyersSquibb (Princeton, NJ), now provides acoordinated monitoring network of morethan 72 medical centers worldwide.

In tandem, several companies haverenewed efforts to discover novel types ofantibiotics to replace the declining numberof agents available to effectively fight resis-tant strains (see Table 1).

Variations on an old themeOne way to diversify the range of antibioticstructures available is to apply combinatorialapproaches to bacterial antibiotic enzymesand pathways. TerraGen Discovery(Vancouver, BC), for example, is scouringenvironmental samples such as soil for organ-isms with novel antimicrobial activities. Byisolating very large segments (up to 300,000bp) of DNA from nonculturable bacteria andthen cloning them into streptomycete bacteria,the company hopes to identify novel antimi-crobials by screening against target bacteria.This approach makes available a huge previ-ously untapped source of natural antibiotics,as only about 1% of microorganisms in anyenvironmental sample can be cultured usingexisting methods. The ability to introduce verylarge DNA segments allows TerraGen to iden-tify novel substances encoded by entire path-ways of enzymes. Recent collaborative agree-ments with the Schering-Plough ResearchInstitute (Kenilworth, NJ) and WarnerLambert/Parke Davis (Ann Arbor, MI) suggestthat the approach has been fruitful.

TerraGen is also exploring the use ofdirect evolution of streptomycete polyketideenzymes to yield novel structures. Polyketides(e.g., erythromycin) are complex organicmolecules synthesized by as many as 50 dif-ferent streptomycete enzymes and carrierproteins from acyl-coenzyme A subunits. InMarch, the company acquired ChromaXome(San Diego, CA), a subsidiary of TregaBiosciences (San Diego, CA), for $6.5 million.ChromaXome has collaborated with BristolMyers Squibb to evolve in vitro polyketidebiosynthesis pathways in streptomycetes. Byconstructing cosmid libraries containing ∼ 40kbp iterative type II polyketide synthase(PKS) gene clusters and associated genes, andsubsequently digesting and randomly religat-ing the products, the company claims it canobtain new structures with probabilities ashigh as 3% (ref. 3).

Elsewhere, Kosan Biosciences (Hayward,CA) is also applying the principles of in vitroDNA engineering to another class of polyke-tide enzymes: the modular type I PKSs. Bymixing and matching enzyme domains fromdiffrent polyketide genes, it recently showedan elegant approach for generating a libraryof more than 50 novel erythromycinmacrolides. This work “suggests that combi-natorial mutations are relatively well tolerat-ed by PKS [domains],” says Kosan’s RobertMcDaniel. Efforts are currently underway tofind more efficient ways of engineeringdiversity and to investigate novel combina-tions of other PKS genes. Indeed, the compa-ny is currently collaborating with Johnson &Johnson (New Brunswick, NJ) to developnew macrolide antibiotics.

Know your enemySimilar to traditional antibiotic discovery,Kosan and TerraGen rely on empiricalscreening to identify agents with bactericidalactivity, without prior knowledge of the tar-get. Many other companies, however, areusing target-directed screens and rationaldrug design to develop new classes of antiin-fectives that extend the range of targetsbeyond those of existing antibiotics. In thisrespect, microbial genomics is revealing aplethora of potential drug targets throughwhole-genome homology searches and func-tional screens (see “Targeting microbialgenomics”). Once these molecules have beenidentified, precise atomic-level informationcan be used to tailor target-specific drugs,accelerating the drug development process.

In collaboration with the NationalResearch Council of Canada (Toronto, ON),GLYCODesign (Toronto, ON) has alreadyfound targets: specific glycosyltransferasesthat play a crucial role in the synthesis ofbacterial lipooligosaccharides (LOS) on thesurface of Neisseria gonorrhoeae and N.meningitidis (the causative agents of gonor-

rhea and meningitis, respectively).Meningococcal LOS is a critical virulencefactor involved in colonization, immuneinvasion, and inflamatory events associatedwith morbidity and mortality. According toDale Cumming, vice president of discovery,mutants of N. gonorrhoeae with truncatedLOS can neither colonize the host nor causedisease, suggesting they are rapidly clearedby the immune system. UsingGLYCODesign’s expertise in developingsmall molecule inhibitors of glycosyltrans-ferases and hydrolases, the collaboration isseeking to develop a novel class of orallyavailable antibiotics.

Another early-stage company adoptingstructure-based drug design of antimicrobialsis Althexis (Waltham, MA). In March,Croatian pharmaceutical company Pliva(Zagreb) invested $14.4 million in the ven-ture, which has as its scientific advisors x-raycrystallographer Manuel Navia and enzymol-ogist Patrick Connelly, formerly of VertexPharmaceuticals’ (Cambridge, MA) HIV pro-tease inhibitor program. As yet, no details ofAlthexis targets have been forthcoming.

Breaking off the attachmentSince bacterial virulence and host range aredetermined by the capacity to attach to specifichost cells, several companies are also searchingfor ways of inhibiting the adhesion process.Not only are nonadherent bacteria easily sweptaway by body fluids like mucosal secretions orurine, but free-floating bacteria are also bettertargets for the immune system or coadminis-tered conventional antibiotics. Stephen Roth,CEO and lead scientist at Neose Technologies(Horsham, PA), claims that natural immunemechanisms provide compelling evidence forthe power of the approach: “Breast milk does-n’t contain bactericidal or bacteriostatic com-pounds,” he says. “All of the antibacterial. ..agents in breast milk are antiadhesives.”

Thus, Neose is developing oligosaccharide

The rise of bacterial resistance

The success of antibiotics has been both a blessing and a curse. On the one hand, theyhave all but eradicated bacterial infectious diseases that once ravaged humankind; on theother, their indiscriminate use has created selection pressure for multiresistant pathogens.A particular concern is the pervasive use of human therapeutics as feed additives forpromoting growth and preventing infections (more than half a million kilograms of β-lactams, tetracyclines, aminoglycosides, and macrolides alone were used in UK agriculturein 1996)8. This has promoted both the exchange of antibiotic-resistance genes on plasmidsor transposons and the spontaneous mutation of drug targets. Disturbingly, recentevidence suggests the predilection of resistant bacteria to develop additional mutations tocompensate for lack of fitness can also perpetuate resistance. To date, bacteria haveevolved a plethora of resistance mechanisms to foil antibiotics, including reduced druguptake into the cell, active efflux of the drug from the cell, modification of antibiotic target toreduce binding, inactivation of antibiotic by enzymatic modification, sequestration ofantibiotic by protein binding, metabolic bypass of the inhibited reaction, binding of specificimmunity protein to the antibiotic, and overproduction of the antibiotic target9. HB

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agents that mimic host cell glycoproteins andglycolipids targeted for attachment by bacte-ria. Because the only way for a bacterial cell tothwart this approach is to change the struc-ture of the adhesin protein—thereby losingits ability to recognize host cells—Roth thinksthat bacterial resistance is unlikely to occur.The company is currently testing twooligosaccharides in phase II clinical trials:NE0080 for the treatment of Helicobacterpylori infections, and NE1530 as a prophylac-tic agent to prevent ear infections in youngchildren caused by Streptococcus pneumoniae,Moraxella catarrhalis, or Haemophilusinfluenzae. Both agents are given as singlemolecules to prevent immune or toxic reac-tions by the patient, although Roth believesthat linking a number of oligosaccharides to aflexible backbone molecule would increasethe approach’s effectiveness.

SIGA Pharmaceuticals (New York, NY),on the other hand, is targeting the attach-ment mechanisms on the bacteria them-selves—in particular, the assembly of piliorganelles (structures that protrude from thebacterial surface and display adhesins) inGram-negative bacteria and specific sortasesused to anchor proteins critical for tissueadherence in the cell wall of Gram-positives.As mutations of critical enzymes in thesepathways disable the ability of bacteria toinvade host tissue, SIGA and collaboratorsVincent Fischetti of Rockefeller University(New York, NY) and Scott Hultgren ofWashington University (St. Louis, MO)believe they could be promising targets.Accordingly, agents are under developmentagainst a chaperone involved in pilus assem-bly in Escherichia coli, H. influenzae, orKlebsiella pneumoniae, and a sortase thatsorts and anchors surface proteins into themembrane of Staphylococcus.

A last approach is to exploit the immunesystem’s own ability to recognize and destroybacterial adhesion proteins through antibodyneutralization. Along these lines,Inhibitex (Atlanta, GA) is usingdomains of microbial surfacecomponent-recognizing adhesive-matrix molecules (MSCRAMMs)on S. aureus and S. epidermis asvaccines for active immunization.The company has also isolatedpolyclonal human antibodies andis developing humanized mono-clonal antibodies againstMSCRAMMs; both agents haveshown effectiveness in passiveimmunization. Clinical trials withantibodies and vaccines areplanned over the next two yearsfor staphylococcal infections inhemodialysis patients. Accordingto Joseph Patti, Inhibitex’s chiefscientific officer, active as well as

passive immunization could be used to pro-tect at-risk surgery patients or immunocom-promised individuals.

Elsewhere, MedImmune (Gaithersburg,MD) is developing vaccines based onFimC–FimH, a complex between the FimCchaperone and the FimH adhesin thatappends pili in uropathogenic E. coli4.According to the company, immunization ofa mouse model with FimH alone reducedbacterial colonization of the bladder mucosaby more than 99% and prevented bacteriafrom infecting the kidneys. In 1998, a vacci-nation study with FimH in nonhuman pri-mates also demonstrated a dramatic decreasein bladder colonization by E. coli in vaccinat-ed animals. The company now plans to startclinical trials using FimC–FimH as a vaccineagainst urinary tract infections with E. coli.

Attacking the bacterial membraneAnother class of agents attracting considerableinterest is the cationic peptides. These mole-cules can be categorized into three main class-es: low-molecular-weight linear peptides (e.g.,cecropin, magainin, and bactenecin); disul-fide-containing peptides (e.g., defensins,tachyplesins, protegrins, attacins, bacterialpermeability-increasing, or BPI, protein, andlysozyme); and serprocidins (e.g., proteinase3, azurocidin, and cathepsin G). They areubiquitous in nature (>300 peptides areknown), evolving as a first-line defense mech-anism among others in plants, insects, crus-taceans, frogs, pigs, cows, and humans3,5.

Although their mechanism of action is stillunclear, they are thought to attach to bacteriallipopolysaccharides and subsequently migratethrough the cell wall to the cytoplasmic mem-brane, where they form channels that disruptthe transmembrane proton gradient, energygeneration and solute transport, and ultimate-ly lead to death. Selectivity for bacteria resultsfrom the low cholesterol content, rich anioniclipid content, and distinctive electrochemical

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gradients of prokaryotic membranes.Most of the cationic peptides have broad-

spectrum activity, although bactenecin,attacin, and BPI target only Gram-negativebacteria. A particular challenge to companiesis the highly toxic nature of the peptides toprokaryotic expression systems.

Magainin Pharmaceuticals (PlymouthMeeting, PA) was quick to recognize thepotential of cationic peptides in antibacterialtreatment. When founder Michael Zasloffobserved that Xenopus frogs appeared resistantto infection after being cut open to removeeggs, a closer inspection revealed skin-secretedpeptides with broad antimicrobial activity,which he coined magainins. Zasloff and histeam have since used magainin sequence infor-mation to tailor drug candidates that are easierto synthesize, have an increased spectrum ofactivity (>3000 clinical isolates), and exhibitenhanced chemical stability. One of these com-pounds, the 22-amino acid peptide pexigananacetate, is the active ingredient in Magainin’sLocilex, a topical cream that just completedphase III trials for the treatment of multior-ganism infections of diabetic foot ulcers. Trialresults demonstrated that the peptide was atleast as effective as conventional antibiotics,but did not show it was significantly better. Asa result, the FDA refused approval in July. Thecompany is now negotiating with its marketingpartner, SmithKline Beecham (King of Prussia,PA), to conduct additional trials against place-bo, as requested by the FDA.

Another company, XOMA (Santa Monica,CA) is also turning its attention to cationicpeptides. In 1991, it licensed the human disul-fide-containing peptide BPI from codiscover-ers Peter Elsbach and Jerrold Weiss of NewYork University (NY). This peptide, whichXOMA now produces recombinantly in-house using Chinese hamster ovary cells, hasthe advantage not only of killing multiresis-tant pathogens, but also of bindinglipopolysaccharide, thus ameliorating the

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Table 1. Selected companies with products in clinic development.

Company Product Status

Cubist Pharmaceuticals Membrane-perforating polyanionic Phase III(Cambridge, MA) peptide daptomycin against S.

pneumoniae and S. aureusInhibitex (Alpharetta, GA) Vaccination with bacterial MSCRAMMs Phase IIIntrabiotics Pharmaceuticals Protegrin analogs against oral mucositis Phase II

(Sunnyvale, CA) due to Candida albicans. Ramoplanin lipopeptide for use against vancomycin-resistant enterococci

Magainin Pharmaceuticals Pexiganan, a cationic peptide from Phase III completed(Plymouth Meeting, PA) frog skin

Micrologix Biotech 12-Amino acid analog of unspecified Phase II(Vancouver, BC, Canada) mammalian cationic peptide against blood-

stream infection in catheterized patientsNeose Technologies (Hosham, PA) Oligosaccharides that mimic host binding sites Phase II

for H. pylori or S. pneumoniae adhesinsXOMA Corporation Cationic peptide BPI for use against Phase III

(Santa Monica, CA) Neisseria meningitidis

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toxic effects of Gram-negative bacteria. InAugust, the company completed phase III tri-als of BPI (Neuprex) for the treatment ofNeisseria meningitidis infections in youngchildren, and the data are currently being pre-pared for review by the FDA. In the mean-time, XOMA has discontinued BPI in anotherindication (bacterial infections after severetrauma and blood loss) and licensed the mol-ecule to Allergan (Irvine, CA) for the develop-ment of medications against eye infections.

Elsewhere, Demegen (Durham, NC) isdeveloping a set of synthetic peptides on thebasis of the structure and mechanism of vari-ous natural molecules. According to vice pres-ident of research, Jesse Jaynes, the companyhas been collating information (e.g., surfacearea, hydrophobicity, volume, charge) onmore than 50 natural antibacterial peptidesthat can be used to predict antimicrobial activ-

ity of synthetic peptides. In time-kill assays,these agents were effective against several hun-dred clinical isolates, including multidrug-resistant S. aureus and Pseudomonas aerugi-nosa. Within the next six months, phase I trialsare planned for the treatment of sexuallytransmitted disease and of burn infections.

In September, FDA also granted fast-track status to a 12–amino acid analog of amammalian cationic peptide (as yet unspeci-fied) manufactured by Micrologix Biotech(Vancouver, BC). The two-part phase II trialsare intended to assess the safety, pharmaco-kinetics, and immunogenicity of the peptidein preventing bloodstream infections incatheterized patients. To produce the toxiccompound in bacteria, Micrologix has devel-oped expression systems that coexpress thecationic peptides with negatively chargedpeptides that can subsequently be removed

by cleavage. The company also has a series ofhybrid peptides based on the cationic pep-tides cecropin (from the giant silk moth) andmelittin (from bee venom). Products areanticipated either as stand-alone bactolysins(lethal) or as enhancins to potentiate theactivity of conventional antibiotics.

Analogs of the human protegrin cationicpeptide are the focus of IntraBioticsPharmaceuticals (Mountain View, CA). InAugust, IntraBiotics announced phase II trialresults in which protegrin IB-367 was effec-tive at reducing oral mucositis due toCandida albicans in patients receiving mye-loablative chemotherapy. The company isalso continuing development of a novelcyclic lipopeptide, Ramoplanin, after a phaseII trial showed efficacy against infections dueto vancomycin-resistant enterococci.

Though not cationic in nature, the lactic

Table 2. Genome projects for some medically important microbes.

Organism Genome size Principal investigators Disease Status(Mbp)

Borellia burgdorferi 1.3 TIGR (Rockville, MD) Lyme disease Completed 12/97Chlamydia pneumoniae 1.2 U. of California and TIGR Respiratory tract infections Completed 5/98Chlamydia trachomatis 1.7 U. of California and TIGR Urinary tract infections Completed 12/98Enterococcus faecalis 3.0 TIGR Wound, respiratory, and urogenital Near completion

tract infection, meningitisEscherichia coli 4.6 U. of Wisconsin Urinary tract and gastrointestinal Completed 9/97

tract infectionsHaemophilus influenzae 1.8 TIGR Meningitis Completed 7/95Helicobacter pylori 1.6 Astra Research (Boston, MA) Gastrointestinal tract infections Completed 2/99Mycobacterium tuberculosis 4.4 Sanger Centre Tuberculosis Completed 6/98

(Cambridge, UK) and TIGRMycoplasma pneumoniae 0.6 TIGR Atypical pneumonia Completed 11/96Neisseria gonorrhoeae 2.2 U. of Oklahoma Urogenital tract infections 98% completeNeisseria meningitidis 2.2 Sanger Centre Meningitis Finished, unpublishedSalmonella typhi 4.5 Sanger Centre Food poisoning Finished, closing gapsSalmonella typhimurium 4.5 Washington U. Food poisoning UnderwayStaphylococcus aureus 2.8 U. of Oklahoma and TIGR Wound infection Finished, closing gapsStreptococcus pneumoniae 2.2 U. of Alabama Meningitis, sinusitis Near completionStreptococcus pyogenes 1.9 U. Oklahoma Throat infection, diarrhea Finished, closing gapsTreponema pallidum 1.1 U. of Texas, and TIGR Syphilis Completed 3/98Ureaplasma urealyticum 0.75 U. of Alabama Urinary tract infections Near completionVibrio cholerae 2.5 TIGR Cholera Near completion

Targeting microbial genomicsA sea change in antibiotics research has resulted from theintroduction of genomics technologies to discovery programs.Microbial genomics provides insights into the way microbesinteract with their hosts and with the drugs used to treat them. Mostimportantly it is revealing new microbial targets for developingentirely new classes of antimicrobial agents. Since 1995, Astra AB(Sweden), Schering-Plough (New York, NY), Roche (Nutley, NJ),Zeneca (Oxford, UK), Pharmacia & UpJohn (Kalamazoo, MI),Abbott (Deerfield, IL), Lilly, Bayer, Bristol Myers Squibb, SmithKlineBeecham, and Monsanto (St. Louis, MO) have all signed deals togain access to microbial sequence information from genomicscompanies, such as Genome Therapeutics (Waltham, MA), HumanGenome Sciences (Rockville, MD), and Incyte Pharmaceuticals(Palo Alto, CA). Publicly funded efforts are also underway at theSanger Center (Cambridge, UK) and the Institute for GenomicResearch (Rockville, MD). The US National Institute of Allergy andInfectious Diseases (Bethesda, MD) and the Wellcome Trust

(London) are also funding microbial sequence projects (e.g., seeTable 2). Once genomes have been sequenced, homologysearches can be carried out to identify putative targets;microarrays can be used to analyze the expression of genes inresponse to infection or drug treatment6. Even before sequence isavailable, genome-tagging approaches allow the identification ofgenes associated with virulence or host invasion7. Indeed, Pfizer(New York, NY) is collaborating with Microcide Pharmaceuticalsto exploit the latter’s method for identifying the 5–10% of genesessential for S. aureus survival. To cite just one example of thepower of genomics, more than 19 new bacterial tRNA synthetasetargets have been identified through the new technologies,according to SmithKline’s Rosenberg (only two were knownbefore 1995). In the next decade, DNA chip technologies alsopromise to transform bacterial diagnostics and tracking of theemergence of resistant strains, enabling the wider use of narrowspectrum agents. HB and Andew Marshall

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acid bacteria antibiotic peptide nisin, devel-oped by AMBI (Tarrytown, NY), has alsocompleted phase I trials. According to thecompany, the program is temporarily onhold while development partners are sought.

In 1997, Cubist Pharmaceuticals(Cambridge, MA) also licensed from Eli Lilly(Indianapolis) daptomycin, a cyclic13–amino acid peptide with a decanoyl sidechain. At the time, Lilly discontinued devel-opment based on the preliminary safety pro-file. “We feel Lilly made a strategic desicionwith inadequate data,” says Francis Tally,Cubist’s vice president for scientific affairs. Hebelieves that toxicity can be avoided by pro-viding once-a-day dosing of the medication.Results from Cubist’s phase III trials testingthe molecule against life-threatening infec-tions by S. aureus and enterococci are expect-ed at the end of this 2000.

More exotic approachesApart from peptides, antibodies, or oligosac-charides, some rather more experimentalstrategies are also being explored as antimi-crobial therapies.

OligoTherapeutics (Wilsonville, OR) ispursuing antisense as an approach tocounter multidrug-resistant bacteria. Thecompany’s vice president, Amy Arrow, saysthat these nucleic acid–based antibiotics(quaintly termed “nubiotics”) are effective invitro against more than 200 clinical mul-tidrug isolates, including vancomycin- andmethicillin-resistant S. aureus.

One advantage of the approach is thatantisense can be easily adapted to accommo-date mutations in bacterial genes encodingtargets. Arrow also counters claims thatoligonucleotides are not taken up by bacteriain vivo, citing a mouse burn model in whichantisense protected 93% of animals fromfatal infection with Pseudomonas. Althoughthe company is searching for developmentpartners, Arrow says its oligonucleotide syn-thesis business is sufficiently profitable tofund trials independently.

Rather than pursuing a nascent technolo-gy, Exponential Biotherapies (PortWashington, NY) has resurrected an experi-mental treatment from the past.Bacteriophage therapy, first investigated inthe 1920s, aims to kill multidrug-resistantbacteria through the phage lytic cycle.

Cooperating with Carl Merril and SankarAdya of the US National Institutes of Health(Bethesda, MD), Exponential has alreadyovercome the first obstacle in phage thera-py—the rapid uptake and inactivation ofphages by the spleen. By injecting phagepreparations into mice, isolating particlesthat remain in circulation, and repeating theprocess iteratively, the company has isolateda strain of phage with a single point muta-tion in the major head protein that is 63,000-

fold better than the wild type at persisting inthe circulation. Exponential has alsoimproved techniques to cleanse phage prepa-rations of bacterial debris—previously amajor problem as bacterial endotoxins cancause life-threatening septic shock. Withthese improvements in hand, companyfounder Richard Carlton plans phase 1 testsearly next year for vancomycin-resistantEnterococcus faecium infections.

Although antibodies would rapidly devel-op against phages following repeated use,Carlton envisions most hospital infectionswould require a single course of treatment.He sees two major advantages for phage ther-apy in life-threatening situations. First,phages are by nature narrow-spectrumagents, which kill only their target strain andspare harmless bacteria, thus preventing theovergrowth of pathogens. Second, phages canadapt quickly to bacterial resistance mecha-nisms, as they also have the ability to mutate.

A spectrum of opportunitiesMost experts agree that a move away fromdrugs like broad-spectrum antibiotics suchas penicillin to more narrow-spectrumagents would represent a small but signifi-cant step in improving the present situation.As Tufts’ Stuart Levy comments, broad-spec-trum agents induce resistance “not only inthe target pathogen, but in other organismsas well.” This markedly increases the chanceof the evolution of novel resistance mecha-nisms and their exchange between diversebacterial populations.

According to Martin Rosenberg, seniorvice president of antiinfectives research atSmithKline, the problem with narrow-spec-trum antibiotics is that diagnostic tests cur-rently require between 36 and 72 h in mosthospitals—too long to delay treatmentchoice in life-threatening infections. What’smore, he believes that rapid diagnosticagents (see “Targeting microbial genomics)are still “a decade away.” Evidently, othersolutions must be found in the meantime.

Of the novel treatments under develop-ment, antiattachment approaches offer theadvantage of preventing adhesion of invadingbacteria and thus increasing exposure toimmune cells. They also decrease the selectivepressure on bacteria to evolve defense strate-gies because the compounds are neither bac-tericidal nor bacteriostatic. Rosenberg cau-tions that these agents may not be useful incuring late-stage infections, however, wherethe pathogen has already colonized the host.

Cationic peptides may also be effectivebecause a complete overhaul of the bacterialsurface structure is required to prevent inter-action of the peptides with the prokaryoticcytoplasmic membrane. As this would need atleast two or three parallel mutations (com-pared with one mutation to become resistant

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against conventional antibiotics), the probabil-ity of resistance is significantly lower. What’smore, the quick and crude way these agents killbacteria could also prevent resistance.

As Magainin’s Zasloff explains, resistanceto cationic peptides is unlikely simplybecause bacteria have not been able to devel-op defense mechanisms in the hundreds ofmillions of years since these peptides evolvedas antimicrobial agents. “The reality is thatwe’ve lived with this system ever since weevolved,” he says, “[and] we exist, in part,because of the continuing efficacy of this sys-tem.” On a more cautionary note, some havequestioned the safety of developing agentslike BPI against bacteria because of their inte-gral role in the body’s own defense mecha-nisms. In this scenario, the emergence ofresistance could lead to the catastrophic cre-ation of “Satan bugs” able to overcome theimmune system of even healthy individuals.

A last challenge is to address the existingculture of indiscriminate and repeated use ofantibiotics both in medicine and agriculture.The problem is that the rules in place do notprevent the abuse and overuse of new antibi-otics. And simple drug usage restrictions bythe government are unlikely to help either. “Assoon as you place restriction on usage, no onewould want to…pay the huge costs of devel-oping such an agent,” says Rosenberg.“Therefore there have to be appropriate incen-tives in the system for creating such reagents.”

Joshua Lederberg of Rockefeller Universitybelieves governments should step in as part-ners for drug companies. “Governments[could] offer subsidies to those companieswho agree to operate under a restrictiveregime,” he suggests. “This would makerestrictions more attractive.” Levy also imag-ines additional ways to provide remunerationfor companies that restrict sales followingapproval. “We should make it economicallyattractive for companies to judiciously markettheir products,” he suggests. “In return, [wecould] extend the patent time or…give themresearch and development costs.”

1. Hancock, R.E.W. & Knowles, D. Are we approachingthe end of the antibiotic era? Curr. Opin. Microbiol.1, 493–494 (1998).

2. http://www.phrma.org/3. Strohl, W.R. (ed.) Biotechnology of antibiotics.

(Marcel Dekker, New York, NY; 1997).4. Choudhury, D. et al. X-ray structure of the FimC–FimH

chaperone–adhesin complex from uropathogenicEscherichia coli. Science 285, 1061–1066 (1999).

5. Ganz T. & Lehrer R.I. Antimicrobial peptides of verte-brates. Curr. Opin. Immunol. 10, 41–44 (1998).

6. Valdovoa, R.H. & Falkow, S. Fluorescence-based iso-lation of bacterial genes expressed within host cells.Science 277, 2007–2011 (1997).

7. Wilson, M. et al. Exploring drug-induced alteration ingene expression in Mycobacterium tuberculosis bymicroarray hybridization. Proc. Natl. Acad. Sci. USA96, 12833–12838 (1999).

8. Davies, J. Inactivation of antibiotics and the dissemina-tion of resistance genes. Science 264, 375–382 (1994)

9. Harvey, J. & Mason, L. The use and misuse of antibi-otics in agriculture. Part 1. Current usage. (SoilAssociation, Bristol, UK; 1998).

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