Current Pharmaceutical Design - Volume 17, Issue 13, 2011

There is a clear and emergent need for a new approach in antimicrobial drug discovery. Pathogenic microorganisms have demonstrated an impressive ability to adapt and develop resistance to antimicrobial agents. The quick emergence of resistance to essentially all broad-spectrum antimicrobial agents has been well established. For example, resistance to penicillin was observed 3 years after its deployment, while the interval was 5 years for tetracycline, 1 year for methicillin, etc. [1]. Moreover, with the possible exception of tigacycline and the narrow spectrum agents linezolid and daptomycin, fluoro-quinolones are the last class of truly broad-spectrum antimicrobial agents with activity against both Gram-positive and Gram-negative bacteria. In this issue of Current Pharmaceutical Design, we outline some of the most exciting developments on the field, with a focus on bacterial and fungal pathogens. First, a series of papers outlines the use of a number of different invertebrate models [2, 3, 4, 5]. The common hypothesis behind the approach outlined in these papers is based on the finding that most pathogens use the same virulence mechanisms when they infect mammals and invertebrates. Our group from Massachusetts General Hospital and Harvard Medical School details the use of the nematode Caenorhabditis elegans as a model host to simultaneously identify new classes of antimicrobial agents (including agents with antivirulence or immunomodulatory efficacy) as well as to evaluate toxicity and efficacy [2]. Du et al. (National University of Singapore) propose to employ the cell-free hemolymph (CFH) from horseshoe crabs as a quick and convenient tool for antimicrobial drug screening [3]. Also, the last two papers in this sub-section outline two important insect models. The paper by Vilcinskas (Justus-Liebig University of Giessen, Giessen, Germany) highlights the use of Galleria mellonella and proposes the development of the insect metalloproteinase inhibitor (IMPI) that exhibits a specific and potent activity against microbial metalloproteinases and inhibits a number of virulence factors of human pathogens [4]. Lionakis et al. summarize the experience at MD Anderson Cancer Center from the use of Drosophila melanogaster, a model host that provides a well studied immune system and powerful genetics and should be especially useful for the study of immunomodulatory compounds [5,6]. Taken together, these surrogate invertebrate hosts fill an important niche in pathogenesis research and provide us with a unique opportunity to identify novel compounds and study basic, evolutionarily conserved aspects of virulence and host response. It should be noted however that invertebrate model systems have different strengths and weaknesses and the selection of a model system dependents on the virulence factors and host responses of interest [7,8]. Findings from invertebrate models should be often validated and studied in mammalian systems. This approach is the basis for the “multi-host” pathogenesis system that is based in cross-species studies among divergent model hosts and allows the discovery of fundamental virulence and host response mechanisms that are independent of the host. An example of this approach is outlined in the paper by Zaborina et al. (University of Chicago). The authors used two widely divergent hosts (the nematode C. elegans and a murine model) to evaluate the hypothesis that the intestinal track of a critically ill host can enhance bacterial virulence. The need for this type of approach is obvious when we consider the ultimate goal of this group that is the development of new agents that can manipulate the “local microenvironment” within the intestine [9]. Importantly, murine models can also be developed further and this issue provides two examples: the intestinal track model by Zaborina et al. noted above and the model by Kerekov et al. (Bulgarian Academy of Sciences) that used severe combined immunodeficiency (SCID) mice to study protein-engineered molecules [10]. In the last sub-section, Termentzi and colleagues (University of Athens, Greece) examine a significant question in drug development: What libraries can provide the highest rate of hits? Especially the complexity and cost associated with the study of natural products and their derivatives can discourage researchers. However, this paper reminds us that “from the 109 new antibacterial drugs approved between 1981 2006, 69% originated from natural products […] while for the antifungal drugs 21% were natural derivatives or compounds mimicing natural products” [11]. Finally, Tegos and colleagues from the University of New Mexico bring everything together as they desribe the microbial multidrug efflux systems and their approaches to discover and develop efflux pump inhibitors [12], while Hamblin and co-workers from the Massachussetts General Hospital report on the drug discovery of antimicrobial photosensitizers using animal models [13]. In conclusion, we envisage that the future of drug discovery will be based on the primary screen of compound libraries in a variety of hosts and we hope that the papers in this issue will inspire researchers to explore new methods for antimicrobial drug discovery. Let's allow our imagination to take us toward novel approaches in drug discovery. These approaches could include the combination of conventional antimicrobial agents with small molecules as well as use of model hosts in the discovery and development of compounds in vivo. Importantly these new systems have the potential to result in the development of less-toxic and more effective antimicrobial agents, including drugs with immunomodulatory and antivirulence activity. The need to redefine our approach could not be more immediate.....

The substantial morbidity and mortality associated with invasive fungal infections constitute undisputed tokens of their severity. The continued expansion of susceptible population groups (such as immunocompromised individuals, patients undergoing extensive surgery, and those hospitalized with serious underlying diseases especially in the intensive care unit) and the limitations of current antifungal agents due to toxicity issues or to the development of resistance, mandate the development of novel antifungal drugs. Currently, drug discovery is transitioning from the traditional in vitro large-scale screens of chemical libraries to more complex bioassays, including in vivo studies on whole animals; invertebrates, such as Caenorhabditis elegans, are thus gaining momentum as screening tools. Key pathogenesis features of fungal infections, including filament formation, are expressed in certain invertebrate and mammalian hosts; among the various potential hosts, C. elegans provides an attractive platform both for the study of host-pathogen interactions and the identification of new antifungal agents. Advantages of compound screening in this facile, relatively inexpensive and not as ethically challenged whole-animal context, include the simultaneous assessment of antifungal efficacy and toxicity that could result in the identification of compounds with distinct mechanisms of action, for example by promoting host immune responses or by impeding fungal virulence factors. With the recent advent of using predictive models to screen for compounds with improved chances of bioavailability in the nematode a priori, high-throughput screening of chemical libraries using the C. elegans-C. albicans antifungal discovery assay holds even greater promise for the identification of novel antifungal agents in the near future.

Horseshoe crabs are an ancient invertebrate which possesses powerful innate immune defense against microbes. The simplicity, specificity and rapidity of its antimicrobial response have accorded the horseshoe crab as an excellent animal model from which immune responsive tissues may be procured for biomedical research. Such usefulness is exemplified by the extensive application for nearly four decades, of the limulus amebocyte lysate (LAL) for sensitive detection of endotoxin in the medical industry. Apart from the amebocytes, the cell-free hemolymph (CFH) of this arthropod offers a large repertoire of evolutionarily conserved proteins, which are highly sensitive to pathogens. This makes the hemolymph an ideal physiological microenvironment for simulating an in vitro infection. We therefore propose to employ the CFH as a quick and convenient tool for antimicrobial drug screening in vitro. This specific drug screening system also provides further optimization of drug design, and selection of drugs with antioxidant properties. Being an easily accessible natural resource, and allowing high-throughput screening with uniform and reliable data output, the horseshoe crab CFH provides a desirable physiological milieu for drug screening and development.

The larvae of the greater wax moth Galleria mellonella prosper in use both as surrogate alternative model hosts for human pathogens and as a whole-animal-high-throughput-system for in vivo testing of antibiotics or mutant-libraries of pathogens. In addition, a broad spectrum of antimicrobial peptides and proteins has been identified in this insect during the past decade among which some appear to be specific for Lepidoptera. Its arsenal of immunity-related effector molecules encompasses peptides and proteins exhibiting potent activity against bacteria, fungi or both, whose potential as new anti-infective therapeutics is presently being explored. Of particular interest is the insect metalloproteinase inhibitor (IMPI) which has been discovered in G. mellonella. The IMPI exhibits a specific and potent activity against thermolysin-like microbial metalloproteinases including a number of prominent virulence and/or pathogenic factors of human pathogens which are responsible for severe symptoms such as septicemia, hemorrhagic tissue bleeding, necrosis and enhancement of vascular permeability. The IMPI and antimicrobial peptides from G. mellonella may provide promising templates for the rational design of new drugs since evidence is available that the combination of antibiotics with inhibitors of pathogen-associated proteolytic enzymes yields synergistic therapeutic effects. The potential and limitations of insect-derived gene-encoded antimicrobial compounds as antiinfective therapeutics are discussed.

The past few decades have seen alarming rates of antimicrobial drug resistance. This trend paralleled a lack of conventional methods of discovery of antibiotics with novel mechanisms of action. Although use of mammalian models remains indispensable for preclinical testing of new antimicrobial compounds, combating emerging multidrug-resistant microbial pathogens may require the use of robust, high-throughput experimental systems that can accelerate drug development. The recent discovery of striking similarities in innate immune signaling pathways between Drosophila melanogaster and mammals has led to a surge in the use of this minihost as an alternative model in studying a variety of infectious diseases. Several genetic screens for microbial pathogenicity in Drosophila identified virulence traits shown to be important for infection in mammals that may serve as targets for future drug development. In addition, conventional antimicrobial agents retain full activity in D. melanogaster infection models, which may pave the way for use of this minihost for high-throughput antimicrobial drug screening. Finally, the availability of genetic tools that allow for conditional inactivation of almost every gene in D. melanogaster is anticipated to result in the discovery of novel immunomodulatory mechanisms of action of newly identified antimicrobial compounds. Overall, the powerful genetics of and capacity for large-scale screening in D. melanogaster make this minihost a promising complementary model that may result in a new paradigm in antimicrobial drug discovery. However, antimicrobial drug discovery in such heterologous, phylogenetically disparate minihosts as the fruit flies, would still require further validation in mammalian models.

The intestinal tract of a host exposed to extreme physiologic stress and modern medical intervention represents a relatively unexplored yet important area of infection research, given the frequency with which this site becomes colonized by highly pathogenic microorganisms that cause subsequent sepsis. Our laboratory has focused on the host tissue derived environmental cues that are released into the intestinal tract during extreme physiologic stress that induce the expression of virulence in colonizing pathogens with the goal of developing novel gut directed therapies that maintain host pathogen neutrality through the course of host stress. Here we demonstrate that maintenance of phosphate sufficiency/ abundance within the intestinal microenvironment may be considered as a universal strategy to prevent virulence activation across a broad range of pathogens that colonize the gut and cause sepsis, given that phosphate depletion occurs following stress and is a universal cue that activates the virulence of a wide variety of organisms. Using small animal models (Caenorhabditis elegans and mice) to create local phosphate depletion at sites of colonization of Pseudomonas aeruginosa, a common cause of lethal gut-derived sepsis, we demonstrate the importance of maintaining phosphate sufficiency to suppress the expression of a lethal phenotype during extreme physiologic stress. The molecular details and potential therapeutic implications are reviewed.

The pathological DNA-specific B cells in Systemic lupus erythematosus are a logical target for a selected therapeutic intervention. It has been recently shown that complement receptor type 1 on human B and T-lymphocytes has suppressive activity. The cocrosslinking of this receptor with the B-cell receptor (BCR) inhibits B cell activation and proliferation and it could be an attractive new target for negative signal delivery. Experimental therapy in humans is limited by many restrictions. Severe combined immunodeficiency (SCID) mice, which lack both T and B lymphocytes and accept xenogenic cells have been used for human cell transfer for evaluating the pathogenesis of human SLE. We hypothesize that it may be possible to re-establish tolerance to native DNA in humanized SCID mice with cells transferred from SLE patients by administering to them a chimeric molecule, containing a monoclonal antibody against human inhibitory complement receptor type 1 coupled to a decapeptide DWEYSVWLSN that mimics DNA antigenically. These protein-engineered molecules are able to cocrosslink selectively the antigen receptors of B-cells possessing anti-native DNA specificity with the inhibitory surface receptors, thus delivering a strong suppressive signal.

Natural products and their derivatives have historically been invaluable as a source of therapeutic agents and have contributed to the discovery of antimicrobial agents. However, today with the development of drug-resistant strains, new scaffolds and new sources of bioactive compounds are needed. To this end, plant derived natural resins are reviewed for their potential application as antimicrobial agents. Natural gums, extracts of the whole resins, as well as specific extracts, fractions, essential oils and isolated compounds from the above resins are discussed in terms of their antifungal, antibacterial, and antiprotozoal activity.

Traditional antimicrobials are increasingly suffering from the emergence of multidrug resistance among pathogenic microorganisms. To overcome these deficiencies, a range of novel approaches to control microbial infections are under investigation as potential alternative treatments. Multidrug efflux is a key target of these efforts. Efflux mechanisms are broadly recognized as major components of resistance to many classes of chemotherapeutic agents as well as antimicrobials. Efflux occurs due to the activity of membrane transporter proteins widely known as Multidrug Efflux Systems (MES). They are implicated in a variety of physiological roles other than efflux and identifying natural substrates and inhibitors is an active and expanding research discipline. One plausible alternative is the combination of conventional antimicrobial agents/antibiotics with small molecules that block MES known as multidrug efflux pump inhibitors (EPIs). An array of approaches in academic and industrial research settings, varying from high-throughput screening (HTS) ventures to bioassay guided purification and determination, have yielded a number of promising EPIs in a series of pathogenic systems. This synergistic discovery platform has been exploited in translational directions beyond the potentiation of conventional antimicrobial treatments. This venture attempts to highlight different tactical elements of this platform, identifying the need for highly informative and comprehensive EPI-discovery strategies. Advances in assay development genomics, proteomics as well as the accumulation of bioactivity and structural information regarding MES facilitates the basis for a new discovery era. This platform is expanding drastically. A combination of chemogenomics and chemoinformatics approaches will integrate data mining with virtual and physical HTS ventures and populate the chemical-biological interface with a plethora of novel chemotypes. This comprehensive step will expedite the preclinical development of lead EPIs.

Antimicrobial photodynamic therapy (aPDT) is an emerging alternative to antibiotics motivated by growing problems with multi-drug resistant pathogens. aPDT uses non-toxic dyes or photosensitizers (PS) in combination with harmless visible of the correct wavelength to be absorbed by the PS. The excited state PS can form a long-lived triplet state that can interact with molecular oxygen to produce reactive oxygen species such as singlet oxygen and hydroxyl radical that kill the microbial cells. To obtain effective PS for treatment of infections it is necessary to use cationic PS with positive charges that are able to bind to and penetrate different classes of microbial cells. Other drug design criteria require PS with high absorption coefficients in the red/near infra-red regions of the spectrum where light penetration into tissue is maximum, high photostability to minimize photobleaching, and devising compounds that will selectively bind to microbial cells rather than host mammalian cells. Several molecular classes fulfill many of these requirements including phenothiazinium dyes, cationic tetrapyrroles such as porphyrins, phthalocyanines and bacteriochlorins, cationic fullerenes and cationic derivatives of other known PS. Larger structures such as conjugates between PS and cationic polymers, cationic nanoparticles and cationic liposomes that contain PS are also effective. In order to demonstrate in vivo efficacy it is necessary to use animal models of localized infections in which both PS and light can be effectively delivered into the infected area. This review will cover a range of mouse models we have developed using bioluminescent pathogens and a sensitive low light imaging system to non-invasively monitor the progress of the infection in real time. Effective aPDT has been demonstrated in acute lethal infections and chronic biofilm infections; in infections caused by Gram-positive, Gram-negative bacteria and fungi; in infections in wounds, third degree burns, skin abrasions and soft-tissue abscesses. This range of animal models also represents a powerful aid in antimicrobial drug discovery.