Combinatorial Chemistry & High Throughput Screening - Volume 8, Issue 3, 2005

Antimicrobial peptides are effectors of innate immunity in phagocytes, body fluids and epithelia. In mammals, defensins, peptides with a characteristic six-cysteine framework, are particularly abundant and widely distributed in various animal species and tissues. The first part of this review provides a historical overview of the ideas that led to the current state-of-the-art in antimicrobial peptides, and the second part is an update on mammalian defensins and their role in host defense to infections.

Antimicrobial peptides (AMPs) are ubiquitous in nature where they play important roles in host defense and microbial control. Despite their natural origin, antimicrobial spectrum and potency, the lead peptide candidates that so far have entered pharmaceutical development have all been further optimized by rational or semi-rational approaches. In recent years, several high throughput screening (HTS) systems have been developed to specifically address optimization of AMPs. These include a range of computational in silico systems and cell-based in vivo systems. The in silico-based screening systems comprise several computational methods such as Quantitative Structure / Activity Relationships (QSAR) as well as simulation methods mimicking peptide / membrane interactions. The in vivo-based systems can be divided in cis-acting and trans-acting screening systems. The cisacting pre-screens, where the AMP exerts its antimicrobial effect on the producing cell, allow screening of millions or even billions of lead candidates for their basic antimicrobial or membrane-perturbating activity. The transacting screens, where the AMP is secreted or actively liberated from the producing cell and interacts with cells different from the producing cell, allow for screening under more complex and application-relevant conditions. This review describes the application of HTS systems employed for AMPs and lists advantages as well as limitations of these systems.

N-Alkylglycine oligomers (peptoids) constitute a family of non-natural peptidomimetics attractive for the early drug discovery process because of their physicochemical features, easy of adaptation to combinatorial chemistry approaches and their proteolytic stability. Consequently, peptoid libraries have found application for discovering hits against a wide diversity of pharmaceutical targets, among which different examples of antibacterials are found. In the present work, research efforts addressed towards the identification of peptoids as antibacterial agents are discussed.

Antibiotic resistant bacterial strains represent a global health problem with a strong social and economic impact. Thus, there is an urgent need for the development of antibiotics with novel mechanisms of action. There is currently an extensive effort to understand the mode of action of antimicrobial peptides which are considered as one alternative to classical antibiotics. The main advantage of this class of substances, when considering bacterial resistance, is that they rapidly, within minutes, kill bacteria. Antimicrobial peptides can be found in every organism and display a wide spectrum of activity. Hence, the goal is to engineer peptides with an improved therapeutic index, i.e. high efficacy and target specificity. For the rational design of such novel antibiotics it is essential to elucidate the molecular mechanism of action. Biophysical studies have been performed using to a large extent membrane model systems demonstrating that there are distinctive different mechanisms of bacterial killing by antimicrobial peptides. One can distinguish between peptides that permeabilize and/or disrupt the bacterial cell membrane and peptides that translocate through the cell membrane and interact with a cytosolic target. Lantibiotics exhibit specific mechanisms, e.g. binding to lipid II, a precursor of the peptidoglycan layer, either resulting in membrane rupture by pore formation or preventing cell wall biosynthesis. The classical models of membrane perturbation, pore formation and carpet mechanism, are discussed and related to other mechanisms that may lead to membrane dysfunction such as formation of lipid-peptide domains or membrane disruption by formation of non-lamellar phases. Emphasis is on the role of membrane lipid composition in these processes and in the translocation of antimicrobial peptides.

Host defense peptides are a vital component of the innate immune systems of humans, other mammals, amphibians, and arthropods. The related cationic antimicrobial peptides are also produced by many species of bacteria and function as part of the antimicrobial arsenal to help the producing organism reduce competition for resources from sensitive species. The antimicrobial activities of many of these peptides have been extensively characterized and the structural requirements for these activities are also becoming increasingly clear. In addition to their known antimicrobial role, many host defense peptides are also involved in a plethora of immune functions in the host. In this review, we examine the role of structure in determining antimicrobial activity of certain prototypical cationic peptides and ways that bacteria have evolved to usurp these activities. We also review recent literature on what structural components are related to these immunomodulatory effects. It must be stressed however that these studies, and the area of peptide research, are still in their infancy.

Cathelicidins are a family of diverse antimicrobial peptides found in granules of mammalian neutrophils. Cathelicidins are active against a broad range of microbes in different environments. Aside from their antimicrobial activity, cathelicidins possess other biological properties including cytotoxic activity towards mammalian cells. Several studies have shown that the amino acid sequence of cathelicidins can be modified to temper undesired properties, such as hemolytic and cytotoxic activity, and at the same time maintain antimicrobial activity. These properties make cathelicidins ideal templates in combinatorial chemistry for designing de novo antimicrobial peptides for therapeutic use. However, one of the major challenges will be to screen these peptides in experimentally relevant models that reflect the environments in which the peptides should be therapeutically active.

Alexander Cole has been investigating two aspects of innate host defense. The first area examines the natural ability of human airway secretions to prevent pathogenic bacterial colonization and aims to identify the host substances that mediate the innate resistance to colonization. His group is examining the host defense of the airways of donors who are healthy and donors who are persistent nasal carriers of Staphylococcus aureus. Their studies to date suggest that a defect exists in the nasal fluid of carriers that permits the colonization of S. aureus in their nasal passages. Ongoing studies to resolve the molecular determinants of S. aureus nasal carriage include a proteomic approach to identify molecules that are dysregulated in S. aureus carrier fluid. Their second focus is based on the reconstruction (from an expressed pseudogene) of a human antimicrobial peptide, called “retrocyclin”, homologous to rhesus monkey circular minidefensins. The peptide had a remarkable ability to inhibit proviral DNA formation and to protect immortalized and primary human CD4+ lymphocytes from in vitro infection by both X4 and R5 strains of HIV-1. Current goals include characterizing retrocyclin's antiretroviral mechanism of action, testing the activity, stability and toxicity of retrocyclin in human fluids, and constructing next-generation analogs for use as antimicrobial therapeutics and preventatives. REPRESENTATIVE PUBLICATIONS 1. Cole, A.M., T. Hong, L.M. Boo, T. Nguyen, C. Zhao, G. Bristol, J.A. Zack, A.J. Waring, O.O. Yang, and R.I. Lehrer. (2002). Retrocyclin: a primate peptide that protects cells from infection by T- and M-tropic strains of HIV-1. Proc. Natl. Acad. Sci. USA 99(4): 1813-1818. 2. Cole, A.M., H-I. Liao, O. Stuchlik, J. Tilan, J. Pohl, and T. Ganz. (2002). Cationic polypeptides are required for antimicrobial activity of human airway fluid. J. Immunol. 169(12): 6985-6991. 3. Ganz, T., V. Gabayan, H.-I. Liao, L. Liu, A. Oren, T. Graf, and A.M. Cole. (2003). Increased pathogenicity of Micrococcus luteus in lysozyme M-deficient mice. Blood 101(6): 2388-2392. 4. Cole, A.M. and R.I. Lehrer. (2003). Minidefensins: antimicrobial peptides with activity against HIV-1. Curr. Pharm. Des. 9(18): 1463-1473. 5. Cole, A.M. (2003). Minidefensins and other antimicrobial peptides: candidate anti-HIV microbicides. Expert Opin. Therapeut. Targets. 7(3): 329-341. 6. Cole, A.M., H-I. Liao, T. Ganz, and O.O. Yang. (2003). Defensin-like antibacterial activity of peptides derived from envelope glycoproteins of HIV-1. FEBS Lett. 535 (1-3): 195-199. 7. Munk, C., G. Wei, O.O. Yang, A.J. Waring, W. Wang, T. Hong, R.I. Lehrer, N.R. Landau and A.M. Cole. (2003). The theta-defensin, Retrocyclin, inhibits HIV-1 entry. AIDS Res. Hum. Retroviruses 19(10): 875-882. 8. Wang, W., S.M. Owen, D.L. Rudolph, A.M. Cole, T. Hong, A.J. Waring, R.B. Lal. and R.I. Lehrer. (2004). Activity of alpha and theta defensins against primary isolates of HIV-1. J. Immunol. 173(1): 515-520. 9. Kalfa, V.C., S.L. Spector, T. Ganz, and A.M. Cole. (2004). Patients with perennial allergic rhinitis and recurrent sinusitis have reduced lysozyme levels in their nasal secretions. Ann. Allergy Asthma Immunol. 93(3): 288-292. 10. Cole, A.M., W. Wang, A.J. Waring, and R.I. Lehrer. (2004). Retrocyclins: using past as prologue. Curr. Prot. & Peptide Sci. 5(5): 373-381.