In Search of Therapy Persister Eradication

Given the prominent role of tolerance in infectious disease, the need for compounds that could eradicate persisters is obvious. Another important factor to consider is the potential causality between tolerance and the rise of resistance. A lengthy, lingering infection that is not eradicated due to its tolerance is likely to provide a fertile ground for producing resistant mutants, or for acquisition of resistance determinants through horizontal transmission from other species. A mathematical model predicts that tolerance substantially increases the danger of resistance (Levin and Rozen 2006). This observation provides an additional incentive for developing compounds that are able to sterilize, rather than merely suppress an infection.

Before we describe possible approaches to eradicating persisters, let us put the problem into perspective and consider the state of affairs in the general field of anti-infective drug discovery. This has been described as a crisis due to an innovation gap (Walsh 2003b). Forty years separate the discovery of the last major class, fluoroquinolones, from the recently introduced linezolid, a narrowspectrum protein synthesis inhibitor acting against Gram-positive bacteria (Zurenko et al. 1997). During this time, pathogens did not take a break, but rapidly acquired resistance to all known antibiotics. There have been several recent successes in addition to linezolid, which include daptomycin (Cubicin, Cubist), a natural product lipopeptide, ramoplanin, a natural product depsipeptide (Genome Therapeutics, currently Oscient), and dalbavancin (Vicuron, currently Pfizer), a molecule related to vancomycin, all acting against Gram-positive species. The recently approved tigecycline (Tigacyl, Wyeth) is a fairly broad-spectrum compound loosely based on tetracycline that is not subject to existing resistances toward the parent antibiotic.

The crisis nonetheless looms, caused by a conspicuous lack of novel classes of broad-spectrum antibiotics in the face of growing drug resistance (Dougherty et al. 2002; Boggs and Miller 2004; Bush 2004; Clardy and Walsh 2004; Projan and Shlaes 2004; Schmid 2004; Silver 2006). The reasons for the crisis are well understood. Bacteria and fungi were the source of natural products, including broad-spectrum compounds, that fueled the golden era of antibiotic discovery during the 1940s and 1950s; Schatz et al. 1944; Demain and Fang 2000; Walsh 2003a). However, culturable microorganisms on which anti-infective drug discovery has been based make up only 0.01%-1% of the total diversity in the environment (Osburne et al. 2000), and this limited resource became overmined.

Synthetic approaches were unable to replace natural products in developing new anti-infective therapies. While it is certainly possible to find synthetic inhibitors against defined bacterial protein targets by screening compound libraries in vitro, attempts to convert such hits into broad-spectrum leads using medicinal chemistry have not been successful. The main obstacle to development of broad-spectrum antibiotics is the penetration barrier of the cell envelope of Gram-negative bacteria. The barrier is multilayered and consists of an inner membrane that restricts the passage of hydrophilic compounds, an outer membrane that restricts penetration of amphipathic compounds (which essentially all drugs are), and MDR pumps that efflux amphipathic substances across the outer membrane. In short, the envelope is designed in a way to restrict penetration of all molecules, while nutrients enter through porins and specialized transporters (Lewis and Lomovskaya 2002; Li and Nikaido 2004). We do not have a rational approach to impart a drug candidate with an ability to cross this barrier, and trial-and-error synthesis does not produce permeating compounds with a measurable degree of probability. As a result, most companies have focused on development of narrow-spectrum antibiotics, or on compounds that are modifications of existing classes. We find ourselves increasingly vulnerable to multidrug-resistant Gram-negative pathogens (Meyer 2005).

Some encouraging developments in solving the antibiotic crisis are worth mentioning. Developing MDR inhibitors is an attractive strategy to produce broad-spectrum dual-compound therapies (Markham and Neyfakh 1996; Hsieh et al. 1998; Markham et al. 1999). Plants use a dual antimicrobial/MDR inhibitor combination to combat their microbial pathogens (Stermitz et al. 2000a). For example, berberine, a weak antimicrobial, is strongly potentiated by 5'-methoxyhydnocarpin, an MDR inhibitor that has no activity on its own. However, presently described plant MDR inhibitors are limited to Gram-positive bacteria, which may explain why there are so few plant pathogens among this group of microorganisms. A synthetic MDR inhibitor active against RND MDRs of Gram-negative species is being developed by Mpex Pharmaceuticals and has entered clinical trials (Lomovskaya and Bostian 2006). NovoBiotic Pharmaceuticals is taking a different approach, based on the assumption that additional classes of broad-spectrum compounds are harbored by the 99% of unculturable microorganisms. Unculturable microorganisms are grown in their natural environment in a diffusion chamber (Kaeberlein et al. 2002) and are then used as a source of secondary metabolites for drug discovery.

An anti-persister drug based on traditional approaches could be produced by combining a conventional antibiotic, such as a fluoroquinolone, and an inhibitor of an essential persister protein. However, it is not clear that inhibition of a single persister protein will be sufficient to have a desired effect. As mentioned above, a mutant in an essential PlsB protein causes E. coli persisters to drop 1-2 log (Spoering et al. 2006), but whether this will be clinically sufficient is not clear.

The requirements for an anti-persister compound are similar to a broad-spectrum antibiotic: it has to penetrate well into cells of both Gram-positive and Gramnegative species. From the above analysis, we can see that the probability of this happening with traditional discovery approaches is 1 drug or less per 40 years. Unlike a conventional antibiotic, however, anti-persister therapy will face an additional hurdle. FDA only requires testing against rapidly growing bacteria, and market conditions are excellent for a new conventional broad-spectrum antibiotic. Why then commit resources to a considerably more challenging anti-persister dual therapy, even if a suitable target will be identified?

In trying to combat persisters, we may have encountered the ultimate adversary. Indeed, persisters evolved through billions of years to perform a single task: survival. During this time, they have encountered all possible harmful compounds, and the inability of any currently employed antibiotics to eliminate persisters provides a sobering view of the magnitude of the challenge. A general recipe in dealing with persisters is then to devise approaches that are not borrowed from and probably do not exist in nature.

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