Project

Design and optimization strategies for synthetic antimicrobial peptoids

Goal: Elucidate physicochemical characteristics of peptoid-based AMP mimics essential for their lipid membrane activity

Date: 26 September 2010

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Konstantin Andreev
added 3 research items
Nature is a major source of inspiration for drug design. Bacteria are developing resistance towards conventional antibiotics. Utilizing antimicrobial peptides (AMPs) – an essential component of innate immune system, as therapeutic agents, may be a viable alternative. Unfortunately, there are a number of serious hurdles on the way towards clinical application of AMPs, including their low bioavailability, costly manufacturing process and toxicity against host cells. To address this issues, current research is focused on the design of synthetic compounds mimicking natural peptides, among which oligo(N-substituted glycines), or peptoids, have shown great promise. Antimicrobial drug efficacy is defined by how it interacts with the membrane of invading pathogen. The physicochemical characteristics of peptoid molecule play a crucial role in these interactions, yet their detailed structure-activity relationships remain obscure. Herein, we have demonstrated that conformational flexibility, cationic charge or hydrophobicity, are critical for oligomeric peptoids to permeate bacterial cell membranes. The outer surface of membrane was modeled by Langmuir monolayers of desired lipid composition and subjected to the constant-pressure insertion assays, epifluorescence microscopy (EFM), synchrotron X-ray reflectivity (XR) and grazing incident-angle X-ray diffraction (GIXD). Our results shed light on the critical details in peptoid mode of action. We believe this will aid in the rational design and of novel anti-infective drugs. Additionally, we have applied our experimental system to model the processes occurring at the air-water interface in lungs. Alveoli are coated by a complex lipid-protein mixture referred to as pulmonary surfactant. This facilitates respiration and prevents alveolar collapse. Patients with respiratory distress receive surfactant replacement therapy that often has the serious drawbacks. X-ray scattering data shows that the structural organization of adsorbed films correlates with surfactant delivery methods onto the respiratory surface. We anticipate that our findings will contribute to the development of novel clinical approaches for treating respiratory diseases.
Konstantin Andreev
added a project goal
Elucidate physicochemical characteristics of peptoid-based AMP mimics essential for their lipid membrane activity
 
Konstantin Andreev
added 5 research items
A promising class of potential new antibiotics are the antimicrobial peptides or their synthetic mimics. Herein we assess the effect of the type of cationic side chain (i.e., guanidino vs. amino groups) on the membrane perturbing mechanism of antimicrobial α-peptide-β-peptoid chimeras. Two separate Langmuir monolayers composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG) and lipopolysaccharide Kdo2-lipid A were applied to model the outer membranes of Gram-positive and Gram-negative bacteria, respectively. We report the results of the measurements using an array of techniques, including high-resolution synchrotron surface X-ray scattering, epifluorescence microscopy, and in vitro antimicrobial activity to study the molecular mechanisms of peptidomimetic interaction with bacterial membranes. We found guanidino group-containing chimeras to exhibit greater disruptive activity on DPPG monolayers than the amino group-containing analogues. However, this effect was not observed for lipopolysaccharide monolayers where the difference was negligible. Furthermore, the addition of the nitrobenzoxadiazole fluorophore did not reduce the insertion activity of these antimicrobials into both model membrane systems examined, which may be useful for future cellular localization studies.
Non-natural oligomeric mimics of antimicrobial peptides (AMPs) can be designed to display chemical moieties analogous to the active side chains of natural peptides, while their abiotic backbone provides protection from proteolytic degradation. N-substituted glycine oligomers (peptoids) are an outstanding example of potential anti-infectious agents that have evoked a significant research effort to optimize their structures. In this study, we evaluated the effect of macrocyclization on the activity of antimicrobial peptoids. Cyclization is effective strategy to restrain the peptoid molecules conformational flexibility. Here, we examined the mode of membrane interactions mode for three pairs of cyclic and linear peptoids using Langmuir monolayer constant pressure insertion assays, fluorescence microscopy, and synchrotron X-ray scattering. The outer leaflets of the outer Gram-negative and cytoplasmic Gram-positive membranes were modeled with LPS (lipid A-kdo2) and DPPG monolayers, respectively. We demonstrate that both cyclic and non-cyclic peptoids readily incorporate into the bacterial membrane mimics, causing a rapid deterioration of the structural ordering of the lipid acyl chains. We also observe that cyclic and linear peptoids differ substantially in their mechanism of action. In particular, analysis of X-ray reflectivity data shows that the cyclic peptoids penetrate into the lipid hydrophobic core to a greater extent than the corresponding linear analogues.
Synthetic polymers mimicking antimicrobial peptides have drawn considerable interest as potential therapeutics. N‐substituted glycines, or peptoids, are recognized by their in vivo stability and ease of synthesis. Peptoids are thought to act primarily on the negatively charged lipids that are abundant in bacterial cell membranes. A mechanistic understanding of lipid–peptoid interaction at the molecular level will provide insights for rational design and optimization of peptoids. Here, we highlight recent studies that utilize synchrotron liquid surface X‐ray scattering to characterize the underlying peptoid interactions with bacterial and eukaryotic membranes. Cellular membranes are highly complex, and difficult to characterize at the molecular level. Model systems including Langmuir monolayers, are used in these studies to reduce system complexity. The general workflow of these systems and the corresponding data analysis techniques are presented alongside recent findings. These studies investigate the role of peptoid physicochemical characteristics on membrane activity. Specifically, the roles of cationic charge, conformational constraint via macrocyclization, and hydrophobicity are shown to correlate their membrane interactions to biological activities in vitro. These structure–activity relationships have led to new insights into the mechanism of action by peptoid antimicrobials, and suggest optimization strategies for future therapeutics based on peptoids.
Konstantin Andreev
added 2 research items
Hydrophobic interactions govern specificity for natural antimicrobial peptides. No such relationship has been established for synthetic peptoids that mimic antimicrobial peptides. Peptoid macrocycles synthesized with five different aromatic groups are investigated by minimum inhibitory and hemolytic concentration assays, epifluorescence microscopy, atomic force microscopy, and X-ray reflectivity. Peptoid hydrophobicity is determined using high performance liquid chromatography. Disruption of bacterial but not eukaryotic lipid membranes is demonstrated on the solid supported lipid bilayers and Langmuir monolayers. X-ray reflectivity studies demonstrate that intercalation of peptoids with zwitterionic or negatively charged lipid membranes is found to be regulated by hydrophobicity. Critical levels of peptoid selectivity are demonstrated and found to be modulated by their hydrophobic groups. It is suggested that peptoids may follow similar optimization schemes as their natural analogues.
Konstantin Andreev
added an update
Konstantin Andreev
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Konstantin Andreev
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Konstantin Andreev
added 2 research items
Synthetic compounds mimicking the structure of natural antimicrobial peptides (AMPs) have a great promise as potential anti-infectious agents due to their stability towards enzymatic degradation, high antibiotic efficiency, and broad adjustability of physicochemical properties. Recently we have demonstrated that antimicrobial activity of AMP synthetic analogs depends on their conformational rigidity. Cyclization is one of the strategies to restrain the flexibility of antimicrobial agents. Herein we present results of a study aimed to establish how the cyclization affects the ability of cyclic N- substituted glycine oligomers (peptoids) to disrupt selectively bacterial, but not mammalian cell, membrane mimics. Lipid monolayers at the air/liquid interface composed of LPS or DPPG were used to model the outer leaflets of Gram-negative and Gram-positive bacterial membranes, respectively, while the DPPC/Cholesterol 6/4 mixed film was used to mimic the mammalian plasma membrane. Interactions of cyclic and linear peptoids with model lipid membranes were investigated using constant-pressure insertion assays, epifluorescence microscopy (EFM), and synchrotron X-ray reflectivity (XR) and grazing incidence X-ray diffraction (GIXD). Insertion assays show that both cyclic and non-cyclic peptoids readily incorporate into the bacterial, but not mammalian, membrane mimics. Moreover their insertion into the bacterial membrane mimics was accompanied by rapid deterioration of the structural order in the lipid acyl chains. Electron density profiles across the film, derived from XR data, demonstrate that both peptoids penetrate into the hydrophobic core of DPPG more efficiently than that of LPS, which might be due to a difference in packing of the hydrophobic core. Nevertheless, our data indicate that, despite these similarities, the mechanisms of action of cyclic and linear peptoids on bacterial membranes are different.