ABSTRACT
Oligomers of acylated lysines (OAKs) are synthetic mimics of host defense peptides (HDPs) with promising antimicrobial properties. Here we challenged the OAK concept for its ability to generate both systemically efficient and economically viable lead compounds for fighting multidrug-resistant bacteria. We describe the design and characterization of a miniature OAK composed of only 3 lysyls and 2 acyls (designated C(12(omega7))K-beta(12)) that preferentially targets gram-positive species by a bacteriostatic mode of action. To gain insight into the mechanism of action, we examined the interaction of OAK with various potential targets, including phospholipid bilayers, using surface plasmon resonance, and Langmuir monolayers, using insertion assays, epifluorescence microscopy, and grazing incidence X-ray diffraction, in a complementary manner. Collectively, the data support the notion that C(12(omega7))K-beta(12) damages the plasma-membrane architecture similarly to HDPs, that is, following a near-classic 2-step interaction including high-affinity electrostatic adhesion and a subsequent shallow insertion that was limited to the phospholipid head group region. Notably, preliminary acute toxicity and efficacy studies performed with mouse models of infection have consolidated the potential of OAK for treating bacterial infections, including systemic treatments of methicillin-resistant Staphylococcus aureus. Such simple yet robust chemicals might be useful for various antibacterial applications while circumventing potential adverse effects associated with cytolytic compounds.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Bacteria/drug effects , Bacterial Infections/drug therapy , Biomimetics , Animals , Fibroblasts/metabolism , Lysine/chemistry , Male , Mice , Mice, Inbred ICR , Microbial Sensitivity Tests , Surface Plasmon Resonance , X-Ray DiffractionABSTRACT
We investigated the potency, selectivity, and mode of action of the oligo-acyl-lysine (OAK) NC(12)-2 beta(12), which was recently suggested to represent the shortest OAK sequence that retains nonhemolytic antibacterial properties. A growth inhibition assay against a panel of 48 bacterial strains confirmed that NC(12)-2 beta(12) exerted potent activity against gram-positive bacteria while exhibiting negligible hemolysis up to at least 100 times the MIC. Interestingly, NC(12)-2 beta(12) demonstrated a bacteriostatic mode of action, unlike previously described OAKs that were bactericidal and essentially active against gram-negative bacteria only. The results of various experiments with binding to model phospholipid membranes correlated well with those of the cytotoxicity experiments and provided a plausible explanation for the observed activity profile. Thus, surface plasmon resonance experiments performed with model bilayers revealed high binding affinity to a membrane composition that mimicked the plasma membrane of staphylococci (global affinity constant [K(app)], 3.7 x 10(6) M(-1)) and significantly lower affinities to mimics of Escherichia coli or red blood cell cytoplasmic membranes. Additional insertion isotherms and epifluorescence microscopy experiments performed with model Langmuir monolayers mimicking the outer leaflet of plasma membranes demonstrated the preferential insertion of NC(12)-2 beta(12) into highly anionic membranes. Finally, we provide mechanistic studies in support of the view that the bacteriostatic effect resulted from a relatively slow process of plasma membrane permeabilization involving discrete leakage of small solutes, such as intracellular ATP. Collectively, the data point to short OAKs as a potential source for new antibacterial compounds that can selectively affect the growth of gram-positive bacteria while circumventing potential adverse effects linked to lytic compounds.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/chemistry , Escherichia coli/drug effects , Gram-Positive Bacteria/drug effects , Anti-Bacterial Agents/chemistry , Cell Membrane/chemistry , Erythrocytes/chemistry , Erythrocytes/drug effects , Escherichia coli/chemistry , Gram-Positive Bacteria/chemistry , Hemolysis/drug effects , Humans , Microscopy, Fluorescence , Staphylococcus/chemistry , Staphylococcus/drug effects , Surface Plasmon ResonanceABSTRACT
Previously characterized chemical mimics of host defense peptides belonging to the oligo-acyl-lysyl (OAK) family have so far failed to demonstrate broad-spectrum antibacterial potency combined with selectivity toward host cells. Here, we investigated OAK sequences and characterized a promising representative, designated C(12)K-3beta(10), with broad-spectrum activity (MIC(90) = 6.2 microM) and low hemotoxicity (LC(50) > 100 microM). Whereas C(12)K-3beta(10) exerted an essentially bactericidal effect, E. coli bacteria were killed faster than S. aureus (minutes versus hours). Mechanistic studies addressing this difference revealed that unlike E. coli, S. aureus bacteria undergo a transient rapid bactericidal stage that over time converts to a bacteriostatic effect. This behavior was dictated by interactions with cell wall-specific components. Preliminary efficacy studies in mice using the thigh infection model demonstrated the OAK's ability to significantly affect bacterial viability upon single-dose systemic treatment (2 mg/kg).
