Design and Characterization of a Broad -Spectrum Bactericidal Acyl-lysyl Oligomer

Helicobacter pylori has developed antimicrobial resistance to virtually all current antibiotics. Thus, there is a pressing need to develop new anti-H. pylori therapies. We recently described a novel oligo-acyl-lysyl (OAK) antimicrobial peptidomimetic, C 12 K-2␤ 12 , that shows potent in vitro bactericidal activity against H. pylori. Herein, we define the mechanism of action and evaluate the in vivo efficacy of C 12 K-2␤ 12 against H. pylori after experimental infection of Mongolian gerbils. We demonstrate using a 1-N-phenylnaphthylamine (fluorescent probe) uptake assay and electron microscopy that C 12 K-2␤ 12 rapidly permeabilizes the bacterial membrane and creates pores that cause bacterial cell lysis. Furthermore, using nucleic acid binding assays, Western blots, and confocal microscopy, we show that C 12 K-2␤ 12 can cross the bacterial membranes into the cytoplasm and tightly bind to bacterial DNA, RNA, and proteins, a property that may result in inhibition of enzymatic activities and macromolecule synthesis. To define the in vivo efficacy of C 12 K-2␤ 12 , H. pylori-infected gerbils were orogastrically treated with increasing doses and concentrations of C 12 K-2␤ 12 1 day or 1 week postinfection. The efficacy of C 12 K-2␤ 12 was strongest in animals that received the largest number of doses at the highest concentration, indicating dose-dependent activity of the peptide (P < 0.001 by analysis of variance [ANOVA]) regardless of the timing of the treatment with C 12 K-2␤ 12 . Overall, our results demonstrate a dual mode of action of C 12 K-2␤ 12 against the H. pylori membrane and cytoplasmic components. Moreover, and consistent with the previously reported in vitro efficacy, C 12 K-2␤ 12 shows significant in vivo efficacy against H. pylori when used as monotherapy. Therefore, OAK peptides may be a valuable resource for therapeutic treatment of H. pylori infection.

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confidence: 99%

The Oligo-Acyl Lysyl Antimicrobial Peptide C ₁₂ K-2β ₁₂ Exhibits a Dual Mechanism of Action and Demonstrates Strong In Vivo Efficacy against Helicobacter pylori

Makobongo

Gancz

Carpenter

et al. 2012

“…Recent designs have, moreover, shown potential for systemic efficacy (4,19,27), including upon coencapsulation with antibiotics in lipid vesicles (7,20). Among these, decanoyl-based OAKs were suggested to represent an efficient platform for the design of improved antibacterial compounds, as illustrated with the broadspectrum bactericidal sequence dodecanoyl-lysyl-[lysyl-aminodecanoyl-lysyl] 3 (C 12 K-3␤ 10 ), which demonstrated potential for systemic treatment of Staphylococcus aureus infection in mice (8). To follow up on these observations, we designed and tested a new series of decanoyl-based derivatives.…”

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confidence: 99%

“…In this respect, simple mimics of HDPs might be helpful in improving understanding of critical mechanistic steps. Among the classes of HDP mimics proposed, OAKs are quite interesting due to their remarkable simplicity.OAKs are composed of a small number of building blocks referred to as ␣ i (acyl-lysyl) or ␤ i (lysyl-acyl-lysyl) subunits (8,13,14), where i specifies the number of carbons in the acyl moiety (Fig. 1).…”

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confidence: 99%

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Antibacterial Properties of an Oligo-Acyl-Lysyl Hexamer Targeting Gram-Negative Species

Zaknoon

Goldberg

Sarig

et al. 2012

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bToward developing new tools for fighting resistance to antibiotics, we investigated the antibacterial properties of a new decanoyl-based oligo-acyl-lysyl (OAK) hexamer, aminododecanoyl-lysyl-[aminodecanoyl-lysyl] 5 (␣ 12 -5␣ 10 ). The OAK exhibited preferential activity against Gram-negative bacteria (GNB), as determined using 36 strains, including diverse species, with an MIC 90 of 6.2 M. The OAK's bactericidal mode of action was associated with rapid membrane depolarization and cell permeabilization, suggesting that the inner membrane was the primary target, whereas the observed binding affinity to lipoteichoic acid suggested that inefficacy against Gram-positive species resulted from a cell wall interaction preventing ␣ 12 -5␣ 10 from reaching internal targets. Interestingly, perturbation of the inner membrane structure and function was preserved at sub-MIC values. This prompted us to assess the OAK's effect on the proton motive force-dependent efflux pump AcrAB-TolC, implicated in the low sensitivity of GNB to various antibiotics, including erythromycin. We found that under sub-MIC conditions, wild-type Escherichia coli was significantly more sensitive to erythromycin (the MIC dropped by >10-fold), unlike its acr-deletion mutant. Collectively, the data suggest a useful approach for treating GNB infections through overcoming antibiotic efflux.H ost defense peptides (HDPs) are part of the innate immune system, providing a first line of defense against a wide range of invading pathogens (3, 28). Current efforts to address the shortcomings of HDPs have focused on biomimetic strategies (6,11,12,22). Several short synthetic oligomers such as polymethacrylate (5), arylamide foldamers (1, 2), and oligo-acyl-lysyls (OAKs) (9) have attracted particular attention due to their lower cost and rapid structural optimization capabilities. Although a number of compounds that demonstrate broad-spectrum antimicrobial activities in vitro have been identified, robust/safe in vivo activity has been a great challenge for most published peptidomimetics. Clearly, to generate efficient HDP mimics, the mechanism of action of this class of molecules needs to be better understood. Many of the details regarding the mechanism of limiting bacterial viability-which can vary widely for different sequences-have yet to be elucidated or are widely debated. Nevertheless, a rich variety of mechanistic studies has emphasized the importance of physicochemical attributes-such as charge and hydrophobicity-for potency and selectivity. In this respect, simple mimics of HDPs might be helpful in improving understanding of critical mechanistic steps. Among the classes of HDP mimics proposed, OAKs are quite interesting due to their remarkable simplicity.OAKs are composed of a small number of building blocks referred to as ␣ i (acyl-lysyl) or ␤ i (lysyl-acyl-lysyl) subunits (8,13,14), where i specifies the number of carbons in the acyl moiety (Fig. 1). Such a design allows systematic and gradual variations of charge and hydrophobicity and the dissection of...

“…Various OAK sequences were shown to affect viability of a wide spectrum of bacterial species (30,38,44,49), while others displayed antiparasite (33, 37) or antitumor (26) activities. In each case, structure-activity relationship (SAR) studies have highlighted two major characteristics: selective cytotoxicity became apparent upon achievement of an optimal hydrophobicity and charge window (30,38,39), whereas poor performances of some of these lipopeptide-like compounds were often linked to the tendency of hydrophobic sequences to aggregate in solution (38,50).Some antibacterial aspects of the OAK sequence C 12 K-7␣ 8 (the molecular structure is shown in Fig. 1) were addressed in previous studies (15,31,39,44).…”

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confidence: 99%

Experimental Conditions That Enhance Potency of an Antibacterial Oligo-Acyl-Lysyl

Goldfeder

Zaknoon

Mor

2010

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Oligo-acyl-lysyls (OAKs) are synthetic mimics of host defense peptides known to exert antibacterial activity both in cultures and in animal models of disease. Here, we investigated how environmental conditions (temperature, pH, and ionic strength) affect the antibacterial properties of an octamer derivative, C 12 K-7␣ 8 . Data obtained with representative bacteria, including the Gram-negative bacterium Escherichia coli and the Gram-positive bacteria Listeria monocytogenes and Staphylococcus aureus, showed that OAK's potency was proportionally affected by pH changes and subsided essentially throughout a wide range of salt concentrations and temperature values, whereas antistaphyloccocal activity was relatively more vulnerable. It was rather the mode of action that was most susceptible to the environmental changes. Thus, OAK's bactericidal effect was limited to a growth-inhibitory effect under acidic pH, low temperatures, or high salt concentrations, whereas basic pH or high temperatures have enhanced the bactericidal kinetics. Properties of binding to model phospholipid membranes provided evidence that correlated the differential modes of action with variable binding affinities. Interestingly, combination of the optimal incubation conditions resulted in a remarkable increase in potency, as expressed by a 16-to 32-fold reduction in the MIC value and by much faster bactericidal rates (>99% death induced within minutes versus hours) compared with the standard incubation conditions. Collectively, the data suggest that OAKs might be useful in developing design strategies for robust antimicrobial peptides that are able to affect a pathogen's viability under a large spectrum of incubation conditions. Host defense peptides (HDPs) are ever-present components of the innate immune system across all organisms (25, 60). These molecules have been widely studied for their vast range of activities, such as antimicrobial, antitumor, mitogenic, and chemical signaling properties (7,22,42,59). Nevertheless, a number of disadvantages, including toxicity, poor bioavailability, and/or high production costs, continue to compromise their potential uses, especially as systemic drugs (6, 22). Also, despite the fact that these compounds have been investigated for over 2 decades, their mechanism of action is not fully understood. Namely, many HDPs are believed to exert antibacterial activity by disrupting the integrity of the cell wall and/or the plasma membrane (16,25,54), whereas others were proposed to hamper intracellular functions (4, 48). While, this nonspecific multitarget mode of action seems likely to significantly prevent the emergence of resistance (36, 59) and indeed significantly accounts for both scientific and applicative widespread interests in these peptides, new innovative tools are needed to shed light onto fine mechanistic details of this complex antimicrobial system.Various synthetic mimics using a small number of building blocks are able to capture the essential antimicrobial properties of HDPs while overcoming some of ...