Challenges for the Development of New Antimicrobials—Rethinking the Approaches: Report of a Workshop

Committee on New Directions in the Study of Antimicrobial Therapeutics: New Classes of Antimicrobials

INTRODUCTION

In 1974, Lewis Thomas described the highest form of medical technology as “the kind that is so effective that it seems to attract the least public notice; it has come to be taken for granted … [and is] exemplified best by … the contemporary use of antibiotics and chemotherapy for bacterial infections … [which] comes as the result of a genuine understanding of disease mechanisms” (Thomas 1974, pp. 34-35). This pronouncement was overoptimistic and premature. Our understanding of the ability of microorganisms to evade modern chemotherapy and to evolve strategies for inactivating our most potent antibiotics was in fact rudimentary, and we are now faced with substantial infectious-disease challenges. In the face of newly emerging infectious organisms, the global crisis in antibiotic resistance, and the threat of bioterrorism, there is a need to invigorate the basic science and technology of anti-infective chemotherapy. To do so, the mechanisms of infectious disease must be better understood, based on a deeper appreciation of microbial physiology, a comprehensive understanding of antibiotic resistance, and a renewed commitment to the discovery of novel antimicrobial molecules and therapies.

There are several indications that new approaches are required to combat emerging infections and the global spread of drug-resistant bacterial pathogens. One is the pattern in rates of death from infectious disease in the 20th century: from 1900 to 1980, the rate dropped from 797 per



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Treating Infectious Diseases in a Microbial World: Report of Two Workshops on Novel Antimicrobial Therapeutics Challenges for the Development of New Antimicrobials—Rethinking the Approaches: Report of a Workshop Committee on New Directions in the Study of Antimicrobial Therapeutics: New Classes of Antimicrobials INTRODUCTION In 1974, Lewis Thomas described the highest form of medical technology as “the kind that is so effective that it seems to attract the least public notice; it has come to be taken for granted … [and is] exemplified best by … the contemporary use of antibiotics and chemotherapy for bacterial infections … [which] comes as the result of a genuine understanding of disease mechanisms” (Thomas 1974, pp. 34-35). This pronouncement was overoptimistic and premature. Our understanding of the ability of microorganisms to evade modern chemotherapy and to evolve strategies for inactivating our most potent antibiotics was in fact rudimentary, and we are now faced with substantial infectious-disease challenges. In the face of newly emerging infectious organisms, the global crisis in antibiotic resistance, and the threat of bioterrorism, there is a need to invigorate the basic science and technology of anti-infective chemotherapy. To do so, the mechanisms of infectious disease must be better understood, based on a deeper appreciation of microbial physiology, a comprehensive understanding of antibiotic resistance, and a renewed commitment to the discovery of novel antimicrobial molecules and therapies. There are several indications that new approaches are required to combat emerging infections and the global spread of drug-resistant bacterial pathogens. One is the pattern in rates of death from infectious disease in the 20th century: from 1900 to 1980, the rate dropped from 797 per

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Treating Infectious Diseases in a Microbial World: Report of Two Workshops on Novel Antimicrobial Therapeutics 100,000 people to 36 per 100,000 people, a reduction by a factor of more than 20 and a testament in part to the efficacy of antibiotics (Armstrong et al. 1999). However, from 1980 to 2000, that rate doubled, largely because of HIV but also due to the spread of drug-resistant bacterial pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci, multiple-drug-resistant gram-negative bacteria, and multiple-drug-resistant tuberculosis (Cohen 2000). While the rise in mortality is due partly to infection in more seriously ill or immunocompromised patients, there is no doubting the need for new strategies and new molecules to treat pathogens that are resistant to nearly the full array of contemporary antibiotics. We are at a critical point, not seen since the pre-antibiotic era, at which infections caused by some bacterial pathogens are untreatable. A second indication of the need for novel antibacterial therapeutics is the almost 40-year innovation gap between introductions of new molecular classes of antibiotics: fluoroquinolones in 1962 and the oxazolidinone linezolid in 2000 (Walsh 2003a,b). A third indication is the recent trend by several large pharmaceutical companies to leave the antibacterial and antifungal therapeutic arenas, suggesting a future decrease in scientific expertise in antibacterial-drug discovery and development skills (Projan 2003; Shlaes 2003). A technology gap is developing and widening, as research on and development of new antimicrobial agents are being de-emphasized or abandoned by many pharmaceutical companies. Treatment of microbial infections—bacterial, fungal, and viral—selects for the emergence of resistant organisms that may be rare in the initial population but become increasingly prevalent under selective drug pressure. In fact, the presence of an antibiotic can accelerate mutation and recombination in bacterial populations and contribute directly to its own obsolescence (Cirz et al. 2005). This is in addition to resistance that may develop outside of the clinical setting; for example, resistance to penicillin had been documented even before its first widespread clinical use (Abraham and Chain 1988). Resistance is prevalent, heritable, and ancient. The need for new generations of anti-infective agents, and in particular new antibacterial agents, is constant, as the emergence of resistance is largely a question of when and not if. Medicinal chemists have been highly successful over the last 50 years in reshaping the scaffolds of earlier antibiotics, both natural and synthetic; for example, current antibiotics include the fourth generation of beta lactams and the third generation of

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Treating Infectious Diseases in a Microbial World: Report of Two Workshops on Novel Antimicrobial Therapeutics macrolides. However, significantly new approaches and strategies for breakthrough molecules have not been forthcoming. Antibiotic resistance affects more than one or a few patients: the global accumulation of resistant bacteria threatens everyone’s health. Once a problem associated only with the sickest patients in intensive-care wards, antibiotic-resistant bacteria have become widespread in communities throughout the world. Resistance genes are not distributed randomly in bacterial populations but are commonly clustered in multiple-drug-resistant strains with resistance spread together. The frequency of international travel, combined with the lack of worldwide standards of antibiotic use, exacerbates the problem. The result is an acceleration of the spread of resistance around the globe and in every environment. All stakeholders recognize that the current antibiotic-resistance crisis is associated with a predictable, inexorable loss of efficacy of our current antimicrobial arsenal, but substantial economic, regulatory, and scientific barriers to the development of new antimicrobial agents and therapies persist (Nathan 2004). This report arises from extensive discussions at a brainstorming workshop organized by the National Research Council of the National Academies under the sponsorship of the National Institute of Allergy and Infectious Diseases. This workshop was planned by the Committee on New Directions in the Study of Antimicrobial Therapeutics: New Classes of Antimicrobials (see Appendix B). Some 40 persons attended the workshop, held on May 23-24, 2005, in Washington, DC, to address strategies for new generations of antimicrobials (see Appendix C for a workshop agenda and participant list). The committee felt that identification of a class of antimicrobials that would be effective in the treatment of a full range of microorganisms—bacteria, viruses, and fungi—without also being detrimental to the host was unlikely. Thus, the workshop was structured around the development of antibiotics that would be effective against bacteria. However, several of the ideas described in this report (e.g., employing natural microbiota to combat pathogens) might also be applicable as treatment strategies against viruses and fungi. The accompanying report on immunomodulation offers additional discussion on treatments that might be effective against the wider range of microorganisms. This report has four major sections: a discussion of the challenge of antibiotic resistance at the population and molecular levels, the importance of understanding bacterial communities and resident microbiota for the discovery of new antimicrobial therapies, consideration of biological processes that can guide strategic approaches to antibiotic development, and

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Treating Infectious Diseases in a Microbial World: Report of Two Workshops on Novel Antimicrobial Therapeutics strategies for discovering new natural and synthetic molecules, including novel screening approaches to bacterial targets. It is hoped that his report will help guide the next decade of antimicrobial research and development. ANTIBIOTIC RESISTANCE Antibiotic Resistance Is Inevitable The goal of the workshop was to identify novel approaches to the development of antimicrobial therapeutics. However, workshop discussions made it clear that even the most innovative antibiotics will be made obsolete, at some point, by the inevitable emergence of resistance. Therefore, the committee concluded that it is worthwhile to identify research that would help to surmount the problem of resistance or at least slow its emergence. The recommendations in this section, although they do not lead directly to the development of novel antibiotics, could be important in increasing the useful lifespan of current and future antibiotics. Bacteria predate humans by billions of years and have evolved a complex series of coping mechanisms that enable them to survive under harsh conditions and in the presence of numerous toxic metabolites. Most antibiotics discovered during the golden age of antibiotics (about 1945-1960) are natural products, produced for the most part by bacteria themselves (Clardy and Walsh 2004). These bioactive secondary metabolites—so called because they are not essential for cell growth or reproduction—may be produced by bacteria to provide a competitive growth advantage by killing susceptible neighbors in the environment, or they may be signaling molecules that have other functions and whose antibiotic activity at high dosages is a side effect. In either case, organisms that produce antibiotics—and organisms that have evolved to live near them—harbor specific and potent resistance strategies that inactivate or otherwise protect them from the antibiotics’ toxic effects. Synthetic antibiotics, such as the fluoroquinolones, would appear to be less susceptible to causing resistance, given that bacteria would not have had millennia of exposure to them. Thus, evolved mechanisms of resistance would be less likely to exist. However, ubiquitous and promiscuous efflux systems have evolved to protect microorganisms from diverse toxic small molecules of natural origin, and these systems often provide cross-protection against such non-natural products. As a result, genes that encode resis-

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Treating Infectious Diseases in a Microbial World: Report of Two Workshops on Novel Antimicrobial Therapeutics tance elements are embedded in the genomes of virtually all bacteria; these hard-wired resistance genes are inherited in vertical fashion, providing continuous protection against toxic agents in a bacterial species even in the absence of prior exposure. Antibiotic-resistance genes, however, are not confined to bacterial genomes. They are also frequently found on mobile genetic elements (plasmids, transposons, and integrons) that readily pass horizontally from organism to organism, even across species boundaries, thereby circumventing the standard parent-to-progeny route of genetic flow (Levy and Marshall 2004). The frequency of selection for such events and for the acquisition of genetic elements increases with increased exposure to antibiotics. It is therefore not surprising that locales in which antibiotic use is rampant—such as hospitals, farms, and child-care settings—are prime sources of organisms that harbor these genetic vehicles. Furthermore, genetic elements passed between organisms in this way often collect several resistance genes; this process is, again, selected for by increased antibiotic use that has given rise to multiple-drug-resistant (MDR) organisms, some of which are untreatable—or nearly so—with the current arsenal of approved drugs. MDR organisms have changed from being primarily a health-care sector problem to being a source of community-acquired disease as patients return to their homes earlier than previously, often remaining on courses of antibiotics. Antibiotic Resistance Is Manageable 1. Predicting Resistance The inevitability of resistance is well accepted by researchers in the field, but there are barriers to collecting and sharing data on resistance among diverse geographic centers and among individual health-care settings within a single geographic region. Efforts to overcome technical and jurisdictional obstacles will improve the ability to monitor resistance, anticipate its spread, and inform health-care practitioners of its existence in the area. Antimicrobial resistance grows as strains of bacteria that carry and exchange resistance genes spread throughout a population or region. Knowledge of resistance in bacteria from prior infections thus helps to target both treatment of new infections and efforts to contain resistance locally and globally as information about recent infections can anticipate antimicrobial

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Treating Infectious Diseases in a Microbial World: Report of Two Workshops on Novel Antimicrobial Therapeutics resistance in new situations. Tens of thousands of clinical and basic research laboratories throughout the world generate resistance data. But very few labs submit these data to appropriate databases that could allow local analysis or linking with a surveillance network. The effectiveness of surveillance data can be enhanced by integrating with other types of information. For instance, molecular studies of resistance can help explain observed resistance phenotypes. Comparison with data on antibiotic usage allows estimation of and potential for the management of antibiotic selection. Data on resistance in non-pathogenic organisms, a potential reservoir of new resistance genes, could help anticipate the emergence of new resistance and to develop predictive diagnostics prospectively. Surveillance of resistance can and should build on existing resources. Clinical laboratories in more than eighty countries have begun to build databases and link them into international networks using free software (WHONET) downloadable from a World Health Organization Web site (O’Brien et al. 2001).1 This type of surveillance data can be complemented and cross-validated by data on isolates collected from clinical laboratories for selected studies in public health reference laboratories or in those supported by pharmaceutical companies. Several companies now collaborate with the Alliance for the Prudent Use of Antibiotics (APUA) to merge their data for these types of meta-analyses (Stelling et al. 2005). Obviously, data in such a database should be as up-to-date as possible and thus able to identify pockets of resistance as they occur. Surveillance needs to be implemented on a grand scale and over a long period to identify trends and provide data for population studies. The surveillance network should not only be nationwide, but linked to international efforts to integrate worldwide data seamlessly. Surveillance should not be limited to the health-care sector. Mechanisms of resistance to any new antibiotic may already exist in nature, so any resistance encountered in nonpathogenic organisms in the environment or antibiotic producers should also be entered into the database. Clinicians and developers of diagnostics would then be aware of resistance mechanisms that may be encountered in the clinic. Such an integrated database would greatly enhance the ability to develop predictive diagnostics that could be rapidly brought on line as resistance elements move around the globe. 1   Available at <http://www.who.int/drugresistance/whonetsoftware> at the time of publication.

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Treating Infectious Diseases in a Microbial World: Report of Two Workshops on Novel Antimicrobial Therapeutics 2. Detecting Resistance Detection of resistance is rooted largely in the century-old technology of growth susceptibility. As a whole, medical microbiology has not adopted state-of-the-art molecular diagnostic measures, and the barriers to gene- or protein-based diagnostics have been substantial. First, in non-sterile sites, such as skin and the gastrointestinal tract, identifying the specific agent causing disease is difficult; even in sterile tissues, such as blood, present-day detection methods are often not sensitive enough to detect disease-causing organisms. Second, the cost associated with molecular tests is often prohibitive. Nevertheless, improved diagnostics could have a revolutionary effect. For example, if a physician could know at the bedside which organism is causing a particular infection and whether that organism is resistant to common antibiotics, treatment could be tailored appropriately. Consequently, antibiotics would be used in a specific fashion, selecting only those likely to be effective; this procedure of judicious and specific antibiotic use would thus help extend the useful lifetime of new antibiotics. Diagnostics able to identify the etiology and antimicrobial susceptibility of all infections could target therapy precisely and eliminate the use of antibacterial agents in patients who do not even have a bacterial infection. If they are done early, such tests could avoid untargeted therapy during the days needed by current diagnostics. In a recent study, polymerase-chain-reaction testing took 6 hours to identify the etiology of 76% of community-acquired pneumonia cases, while older tests took several days to identify 49.5% (Templeton et al. 2005). Specifically, research into diagnostic tests that can reliably and quickly identify pathogenic organisms and their resistance profiles should be encouraged. Development of such tests may be difficult but could lead to significant advancement in the treatment of infectious disease. To effect the greatest reduction in inappropriate antibiotic use, such tests would need to be so rapid and reliable that clinicians would be comfortable waiting for the results before beginning antibiotic treatment. Determining the necessary degree of reliability for these diagnoses is an open challenge as the tests must not only lead to successful diagnosis of the pathogen, but also have the confidence of clinicians. A major issue in resistance is that not only disease-causing organisms, but also other resident organisms and the host itself are exposed to an antibiotic. Minimizing exposure through precise choice of antibiotic is critical

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Treating Infectious Diseases in a Microbial World: Report of Two Workshops on Novel Antimicrobial Therapeutics in preventing the emergence of resistance by reducing selection in off-target organisms. That is, the use of a narrow-spectrum antimicrobial agent optimized for use against the disease-causing organism would be less likely to select for resistance in non-targeted microorganisms. Advanced diagnostics discussed above will facilitate tight targeting of pathogens and thereby enable the productive exploration of target-specific antibiotics. The advances could include selective interruption of organism-specific processes, such as virulence mechanisms, adhesion of surface antigens, and resistance mechanisms. Enhancing the host response at the site of infection is a potential creative approach to activating toxic molecules where they are needed. New tissue-specific delivery vehicles would greatly help to decrease the exposure of non-target species to antibiotics. However, it should be noted that these strategies will not eliminate resistance as resistant microorganisms also arise from the use of antibiotics in non-clinical settings such as agricultural use. 3. Deterring Resistance How resistance elements are selected and spread throughout microbial communities is largely unknown. Understanding the fundamental principles underlying how pathogenic organisms and normal microbiota communicate and exchange genetic information is a key to the ability to manage the spread of resistance. Research on the molecular mechanisms that facilitate resistance is also warranted. For example, do some antimicrobial agents inherently activate mutagenic pathways that can lead to resistance (e.g., Cirz et al. 2005)? If so, are there classes of molecules that are less susceptible to this action? Similarly, are there antimicrobial targets that are less tolerant of mutations selected by the presence of antibiotics? Furthermore, how such issues as antibiotic dosage and scheduling, antibiotic mixtures, and interactions with other drugs affect the emergence of resistance is not well understood. The example of amoxicillin/clavulanate potassium (Augmentin), a highly successful combination of an antibiotic and an inhibitor of resistance, should be emulated (Matti et al. 1998). Combination therapy to inhibit the emergence of resistance has also been used in the treatment of HIV (HAART therapy) and tuberculosis (isoniazid, rifampin, and pyrazinamide) (Finch et al. 2003). Leveraging knowledge of molecular mechanisms of resistance in the development of selective inhibitors has the potential to rescue the activity of proven antibiotics that

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Treating Infectious Diseases in a Microbial World: Report of Two Workshops on Novel Antimicrobial Therapeutics have well-established pharmacological and disease profiles (Wright 2000). Extending the clinical lifetimes of proven antibiotics in this fashion holds great promise. The challenge will be to selectively target the most important resistance mechanisms. Recommendations on Resistance To respond to issues of resistance to antimicrobials, the committee recommends the following research directions and action items: A-1.1 Establishment of a simple and readily searchable antibiotic resistance database into which participating institutions would upload resistance data in real time. A-1.2 New rapid diagnostics to detect pathogens and their resistance to inform therapy in real time. A-1.3 Development of strategies that will selectively target pathogenic organisms while avoiding targeting the host and beneficial or benign organisms. A-1.4 Identifying the sources of resistance mechanisms, their evolution, and the ways in which they are spread in microbial communities, to elucidate the various ways in which resistance can be manifest. A-1.5 Development of strategies that target and selectively block antibiotic resistance mechanisms to rescue antibiotic activity. A-1.6 Exploration of the effect of antibiotic usage, alone and in combination, on the development of resistance. A MICROBIAL COMMUNITY APPROACH TO NEW ANTIBACTERIALS Characterization of Communities of Microbiota There is growing evidence of the important role played by resident microbiota in offering protection from infectious disease. Rather than continuing the traditional approach of killing bacteria wherever they occur, there is a need to develop new antimicrobial strategies aimed at subtle manipulation of bacterial behavior. Such therapies would favor natural host defenses and the maintenance of the normal microbiota to keep growth of

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Treating Infectious Diseases in a Microbial World: Report of Two Workshops on Novel Antimicrobial Therapeutics pathogenic species in check. At the outset, design of strategies for novel antibiotics should include exploration of strategies for exploiting beneficial and commensal bacteria in fighting infections in sites where normal microbiota reside. To develop such therapies, a deeper understanding of the diversity and ecology of the normal human microbiota and how these communities are established and stably maintained is needed. At present, understanding of human microbiota communities and their true diversity and ecology is limited (Eckburg et al. 2005; Hooper and Gordon 2001; Wilson 2005; Nataro et al. 2005). Precise definition of these associations in human health and in disease will allow the development of nontraditional therapeutics aimed at manipulating bacteria and their environment to enhance the maintenance and proliferation of the normal microbiota and inhibit the growth of pathogens. The committee recommends the following to deepen understanding of natural microbiota:2 A-2.1 Characterization and enumeration of the normal resident microbiota in human hosts. A-2.2 Understanding the relationship between resident microbiota populations and human health. Manipulating Bacterial Signaling and Communication The last decade has taught that bacteria do not live independent lives but, rather, communicate within and among species by using a variety of secreted signal molecules (Miller and Bassler 2001). Production, detection and response to these molecules allow bacteria to take a census of the population and synchronize behavior on a population-wide scale. This process, called quorum sensing, is critical for many pathogens because expressing virulence genes as a group ensures that pathogenicity factors are released only when bacterial numbers are sufficient to guarantee success against the host (Donabedian 2003; Williams 2002). More complete understanding of the chemicals that bacteria use for signaling and how bacteria integrate and interpret chemical information in their environment would allow investigation of their use in antibacterial treatments. 2   These recommendations are similar to those in the accompanying report on immunomodulation (e.g., recommendation I-6.1).

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Treating Infectious Diseases in a Microbial World: Report of Two Workshops on Novel Antimicrobial Therapeutics At present, only four predominant classes of molecules used for communication are known: acylhomoserine lactones in gram-negative bacteria (Parsek et al. 1999), oligopeptides in gram-positive bacteria (Lazazzera and Grossman 1998), γ-butyrolactones in the streptomycete subset of gram-positive bacteria (Chater and Horinouchi 2003), and a furanone called AI-2 that is used for signaling in diverse bacterial species (Miller et al. 2004; Chen et al. 2002). The chemical lexicon is probably much larger than is currently recognized, and a continued study of cell-cell signaling with an emphasis on further definition of the chemical moieties used should reveal new classes of molecules that convey information about the community. Manipulation of bacterial cell-cell signaling systems has potential use in novel antimicrobial therapies (Williams 2002; Dong et al. 2001). Enhancing growth-promotion signals of the normal microbiota at the expense of non-indigenous species might restore the normal microbial balanced state. Alternatively, specifically interrupting signaling between pathogens or giving improper signals might cripple the pathogens and make them easier to kill with standard antibiotics or by the immune system. Chemical communication between bacteria is critical for establishing and maintaining complex structured communities, such as biofilms (Davis et al. 1998; Costerton et al. 1994). Disruption of cell-cell signaling systems might provide novel opportunities for antibiotic therapy (Hentzer et al. 2003; Merritt et al. 2003; Ren et al. 2002). Furthermore, it is possible that the host recognizes and responds to bacterial signaling molecules (Chun et al. 2004), and understanding whether and how this occurs could lead to therapies for priming or boosting host defenses. Beyond chemicals used for quorum-sensing cell-cell communication, bacteria make and release a rich variety of compounds, and enormous amounts of information could be encoded in these molecules. Bacteria probably interpret these compounds for important information about the species composition of the environment, the growth conditions, the vitality of the community, and so on. Streptomycetes are known to produce an extraordinary collection of so-called secondary metabolites (Bibb 2005). These chemicals have been and continue to be mined for those with desired activities, such as anticancer properties. The compounds are viewed as a rich storehouse of novel pharmaceuticals, but why are the bacteria making and releasing them? How do the bacteria recognize and respond to the information encoded in these chemicals? Normal human microbiota might not be as prolific in chemical production and release as the streptomycetes, but they also release complex chemical mixtures, and current understand-

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Treating Infectious Diseases in a Microbial World: Report of Two Workshops on Novel Antimicrobial Therapeutics metabolic pathways that are cryptic when producer organisms are grown in standard culture conditions. For example, only three of the predicted 25 polyketide synthase biosynthetic gene clusters in Streptomyces avermitilis have been shown to be active (Omura et al. 2001). Similarly, several sets of antibiotic gene clusters in Bacillus subtilis are expressed only in starvation-induced conditions not generally present in laboratory cultures (Stein 2005). Continued, even increased, attention should be paid to natural strategies used by prokaryotes and eukaryotes against microorganisms. Hosts respond to microbial infection by secretion of peptidic molecules, such as defensins, to act locally. Narrow-spectrum protein toxins (bacteriocins) are a predominant strategy in natural microbial communities for killing neighboring strains; for example, bacteriocidic microcins secreted by one strain of E. coli kill neighboring strains selectively but have minimal effect on the microbial community. This effect—selectively removing pathogens while leaving commensal bacteria unharmed—would be a desired feature of antibiotics. Efforts should be focused on delivery strategies for peptides, bacteriocins, and phage-based lytic proteins (Yoong et al. 2004). Bacteria produce bioactive molecules through a series of biosynthetic steps. The idea behind combinatorial biosynthesis is to break the biosynthetic pathways down into modules and combine the modules in a well-characterized host to generate novel end products. The ability to express biosynthetic-pathway genes in heterologous hosts will be required for efficient combinatorial biosynthesis. Ultimately, full realization of the potential of combinatorial biosynthesis will require engineering bacteria that can make the monomeric building blocks that are required for running the assembly lines and expressing a full range of post-assembly-line tailoring enzymes. Ideally, the ability to shuffle protein domains and modules in the polyketide synthase and nonribosomal peptide synthetase pathways and to engineer intersections with terpenoid and other pathways to merge other chemical frameworks onto polyketide and peptide scaffolds will be needed for maximizing natural diversity (Walsh 2004). Finally, further investigation of the molecular biology underlying bacterial cell death is needed. In most cases, there is little understanding of why bacteria that are susceptible to particular antibiotics die or which traditional targets hold more promise for the development of bactericidal antibiotics. Bacteria, like eukaryotes, may have mechanisms of programmed cell death. If so, the genetic regulatory programs and the biochemical processes associated with the triggering of cell death could provide new targets

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Treating Infectious Diseases in a Microbial World: Report of Two Workshops on Novel Antimicrobial Therapeutics for antibiotic development (Engelberg-Kulka et al. 2004). Current understanding of bacterial cell death lags behind understanding of this process in higher organisms. To maximize the discovery of novel natural molecules and to increase the ability to generate variations on natural molecules, the committee recommends the following: A-6.1 Increased sampling in diverse environments and increased application of the techniques of metagenomics to identify bioactive compounds produced by currently unknown and uncultured microorganisms. A-6.2 The development of novel and varied culture conditions to identify cryptic metabolic pathways in currently cultivated strains. A-6.3 Increased research on the role of host-derived antimicrobial peptides, phage lytic proteins, and bacteriocins in the ecology of host-bacteria interactions to improve delivery strategies for these natural products. A-6.4 Increased research on combinatorial biosynthesis to allow the most varied possible uses of the novel biosynthetic pathways found in known and unknown organisms. A-6.5 Increased research on bacterial cell death, including investigation of programmed cell death and how antibacterials kill to exploit new strategies for the elimination of pathogens. Developing Synthetic Molecules: Diversity, Bioactivity, and Specificity In addition to discovering and elaborating on the bioactive compounds made by bacteria themselves, the design of synthetic antibiotic molecules should also be pursued. The committee identified three kinds of research that would contribute to greater success in the design of synthetic antibiotics: developing techniques that make it easier to generate diversity in synthetic molecules, increasing understanding of the characteristics that allow molecules to enter and remain in cells, and developing the ability to move beyond using growth inhibition as the measure of a compound’s activity and achieving a more sophisticated understanding of how compounds affect metabolism. One of the principles of synthetic-molecule construction is modularity, with variable shape and architecture of modular cores; linkers or con-

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Treating Infectious Diseases in a Microbial World: Report of Two Workshops on Novel Antimicrobial Therapeutics nectors that are also variable in shape, length, and polarity; and surface functional groups in each module that allow rapid elaboration. A goal of such modularity is to build on initial leads and elaborate them in any direction for optimization. A further goal would be to minimize blunt ends—scaffold elements that prevent expansion—in any modular array of synthetic molecules. Two promising techniques for generating greater diversity in synthetic-molecular construction are “click chemistry” and programming small molecules genetically. The “click chemistry” paradigm elaborated by Sharpless and colleagues (Kolb et al. 2001)—such as the coupling of azides and alkynes with copper catalysis in aqueous solution under mild conditions—is a leading example of rapid modular combinatorial chemistry. In favorable cases, the shape of the binding pocket in a target protein can guide covalent couplings into complementarily shaped molecules (Manetsch et al. 2004). The binding of small molecule fragments capable of self-assembly in cavities of target proteins may become a generalizable strategy to produce small-molecule architecture complementary to and with high affinity for target bacterial proteins. Another promising direction for antimicrobial chemical libraries is genetically programmable small molecules (Li and Liu 2004; Halpin et al. 2004; Halpin and Harbury 2004a,b). DNA tethering can allow enhanced adjacency to promote new, high-yield chemistry and the creation of large libraries from which molecules can be selected for function. In a screen for nanomolar binders to a target bacterial protein, 105 promising molecules could be obtained from very large (for example, 1012 entries) programmable libraries; these 100,000 winners could be put through a further series of functional screens (such as for whole-cell activity) with the prospect that there would be many with high activity for further structural optimization. A key advantage of the DNA-directed programmable approach is the opportunity to evolve molecules to optimize a selected function. However, the ability to generate diverse molecules will not be enough. A major bottleneck in drug design and evaluation is the optimization process for turning hits into molecules that will work in the host. What is needed is the ability to move smoothly back and forth between changing chemical structure and activity in the host; computational prediction and rapid preparation of related families of molecules must be integrated with pharmacokinetic measurements. Computational biology and systems biology must become central to the evaluation of new molecular scaffolds in infected animals and humans to predict the safety and efficacy of new classes of molecules.

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Treating Infectious Diseases in a Microbial World: Report of Two Workshops on Novel Antimicrobial Therapeutics To increase the ability to generate synthetic molecules that are not only diverse but also bioactive in a predictable way, the committee recommends the following: A-7.1 Development of small-molecule libraries customized for bacterial targets. A-7.2 Increased research on DNA-directed synthesis of diverse collections of small molecules for screening and selection against bacterial targets. A-7.3 Greater emphasis on projects that systematically relate chemical structure to biological activity. Mining Historical Knowledge Many pharmaceutical and biotechnology companies that have a history of pursuing the identification of new antibiotics probably have files on the development and testing of molecules that could be mined for promising leads. It might also be valuable to interview those who did the work. The workshop and committee members’ experiences have brought forth anecdotes about valuable drugs nearly abandoned because of shifts in corporate policy but then saved by the conviction or special insight of one investigator. Interviews and data mining of now-ignored records might reveal promising molecules on which much work has already been conducted, but that were dropped for reasons unrelated to their efficacy. Other molecules may have encountered obstacles that can now be circumvented by new technology or understanding. Such a historical and biographical approach is unusual in drug development and in biomedical research, but may reinvigorate now-ignored research that showed promise. Screening Issues: How to Find Functional Properties in Candidate Molecules If it becomes possible to generate a multitude of diverse molecules, the next challenge is to improve the ability to screen them for antibiotic potential. Cell-based screening, in which the ability of compounds to kill bacteria growing in ideal, monoculture conditions is tested, has the advantage of identifying compounds with the right physical properties to penetrate and persist in cells (and affect their growth). However, because the targets are unknown, it is difficult to predict and test how the activity of the compounds could be enhanced. In contrast, target-based screening identifies

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Treating Infectious Diseases in a Microbial World: Report of Two Workshops on Novel Antimicrobial Therapeutics compounds that bind to or inhibit a bacterial target that is believed to be essential for bacterial survival. It has proved challenging to endow compounds that have the desired activity with the ability to penetrate cells and reach their targets. Both target- and cell-based approaches have value, but fresh approaches are needed because of the weaknesses inherent in each (Brown and Wright 2005). For example, in cell-based screens, it is important to collect more information beyond the single criterion of growth inhibition so that it will be possible to characterize a molecule’s activity and narrow its possible targets (Schreiber 2005). Screens that provide detailed information about intermediate states of bacterial cell perturbation, including gene chip arrays and metabolome profiling, would be valuable. Validation of these screens on the dozens of existing classes of antibiotics would provide the beginning of a comprehensive database. Inclusion of both pathogenic and commensal bacteria in high-density screening arrays would lend a systems-biology perspective and build the detailed resolution to identify sites of compound action. A few hundred antimicrobial small molecules would become standard probes to provide response patterns with which new candidate molecules could be compared. In target-based screens, greater emphasis needs to be placed on increasing the likelihood of success in cell penetration and persistence of active molecules. The physical-chemical characteristics of cell permeability are poorly understood. Further research is needed to characterize the functional properties of molecules that minimize interaction with bacterial-membrane efflux pumps and allow penetration and persistent accumulation in pathogenic bacteria. The high-density screening techniques developed to improve cell-based screening could also be used to screen for these characteristics. Such efforts may help augment traditional and current chemical libraries to have a greater representation of molecules likely to be successful antibiotics and provide the data to optimize antibiotic-like molecules in future libraries. As an example of the value of combining cell- and target-based approaches in the development of novel antimicrobials, a narrow-spectrum diarylquinolone, acting against mycobacteria by inhibition of the F0 subunit of ATP synthase (Andries et al. 2005), has recently been discovered by medicinal-chemistry optimization in whole-cell killing assays that used the fast-growing M. smegmatis as an initial M. tuberculosis surrogate. The mode of action was determined by whole-genome sequencing of resistant organ-

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Treating Infectious Diseases in a Microbial World: Report of Two Workshops on Novel Antimicrobial Therapeutics isms and then pharmacokinetics in mice optimized before an initial phase I trial for safety and tolerability in humans. For microbial cell-based killing assays as the starting point, this study may become a paradigm of strategic execution. Both screening approaches would benefit from a publicly available collection of molecules that have been shown to have antimicrobial activity. Many such molecules have been reported over the last 6 decades of antibiotic research but may be languishing in the private coffers of companies not actively developing antimicrobials. Molecules could be collected from those companies or resynthesized. Another avenue worth pursuing would be donation of such molecules, especially naturally occurring ones, by pharmaceutical or biotechnology companies. The establishment of a collection of active antimicrobial compounds, numbering in the thousands, would represent a precious archive available to the research community for information-rich screens. Such a collection might also serve as small molecule-based microarrays for target-based screening. Finally, measurements of the efficacy of novel therapeutics must use assays that closely mimic in vivo conditions. Traditional tests for antibiotic effectiveness rely on in vitro assays typically of single-species bacterial cultures grown under standardized conditions in defined laboratory media. These research assays do not effectively mimic the environments that bacteria experience in a host and although positive outcomes reliably occur in the laboratory, they are not always good predictors of outcomes in vivo. Treatment failure due to phenotypic tolerance needs to be examined so that therapies that avoid problems associated with noninherited resistance can be developed. To improve the identification and characterization of bioactive compounds, the committee recommends the following: A-8.1 Development of cell-based screening techniques that collect detailed information on cell metabolism through gene arrays, metabolome profiling, and other measurements. A-8.2 Increased research on the chemical properties necessary for cell permeability and retention. A-8.3 Establishment of a publicly available collection of molecules that have antibiotic activity. A-8.4 Development of new assays that mimic in vivo conditions.

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Treating Infectious Diseases in a Microbial World: Report of Two Workshops on Novel Antimicrobial Therapeutics HOW CAN THIS WORK BE CARRIED OUT? Not only new scientific approaches, but also legislative and institutional actions may be required to improve the prospects for development of new antimicrobial agents and prolongation of their efficacy. The introduction of three bills in the U.S. Congress bearing on antibiotic use and development suggests that major changes in society’s approach to antibiotics may be on the horizon (Nathan and Goldberg 2005). While it was beyond the scope of the committee to consider non-scientific matters, the participants in the workshop noted that the scientific, regulatory and economic aspects of the development of antimicrobials are extensively interconnected (Nathan 2004). REFERENCES Abraham, E.P., and Chain, E. (1988) An enzyme from bacteria able to destroy penicillin. 1940. Rev. Infect. Dis. 10(4), 677-8. Alroy, I., Tuvia, S., Greener, T., Gordon, D., Barr, H.M., Taglicht, D., Mandil-Levin, R., Ben-Avraham, D., Konforty, D., Nir, A., Levius, O., Bicoviski, V., Dori, M., Cohen, S., Yaar, L., Erez, O., Propheta-Meiran, O., Koskas, M., Caspi-Bachar, E., Alchanati, I., Sela-Brown, A., Moskowitz, H., Tessmer, U., Schubert, U., and Reiss, Y. (2005) The trans-Golgi network-associated human ubiquitin-protein ligase POSH is essential for HIV type 1 production. Proc. Natl. Acad. Sci. USA 102, 1478-83. Andries, K., Verhasselt, P., Guillemont, J., Göhlmann, H.W.H., Neefs, J.-M., Winkler, H., Van Gestel, J., Timmerman, P., Zhu, M., Lee, E., Williams, P., de Chaffoy, D., Huitric, E., Hoffner, S., Cambau, E., Truffot-Pernot, C., Lounis, N., and Jarlier, V. (2005) A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science 307, 223-7. Armstrong, G.L., Conn, L.A., and Pinner, R.W. (1999) Trends in infectious disease mortality in the United States during the 20th century. J. Am. Med. Assoc. 281(1), 61-6. Baker, M. (2005) Better living through microbes. Nat. Biotechnol. 23, 645-7. Bibb, M.J. (2005) Regulation of secondary metabolism in streptomycetes. Curr. Opin. Microbiol. 8, 208-15. Bister, B., Bischoff, D., Ströbele, M., Riedlinger, J., Reicke, A., Wolter, F., Bull, A.T., Zähner, H., Fiedler, H.-P., and Süssmuth, R.D. (2004) Abyssomicin C—a polycyclic antibiotic from a marine Verrucosispora strain as an inhibitor of the p-aminobenzoic acid/ tetrahydrofolate biosynthesis pathway. Agnew. Chem. Int. Ed. Engl. 43(19), 2574-6. Boshoff, H.I., Reed, M.B., Barry, C.E., 3rd, and Mizrahi, V. (2003) DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis. Cell 113, 183-93. Brown, E.D., and Wright, G.D. (2005) New targets and screening approaches in antimicrobial drug discovery. Chem. Rev. 105, 759-74.

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