Effective in vivo treatment of acute lung injury with helical, amphipathic peptoid mimics of pulmonary surfactant proteins.

Acute lung injury (ALI) leads to progressive loss of breathing capacity and hypoxemia, as well as pulmonary surfactant dysfunction. ALI's pathogenesis and management are complex, and it is a significant cause of morbidity and mortality worldwide. Exogenous surfactant therapy, even for research purposes, is impractical for adults because of the high cost of current surfactant preparations. Prior in vitro work has shown that poly-N-substituted glycines (peptoids), in a biomimetic lipid mixture, emulate key biophysical activities of lung surfactant proteins B and C at the air-water interface. Here we report good in vivo efficacy of a peptoid-based surfactant, compared with extracted animal surfactant and a synthetic lipid formulation, in a rat model of lavage-induced ALI. Adult rats were subjected to whole-lung lavage followed by administration of surfactant formulations and monitoring of outcomes. Treatment with a surfactant protein C mimic formulation improved blood oxygenation, blood pH, shunt fraction, and peak inspiratory pressure to a greater degree than surfactant protein B mimic or combined formulations. All peptoid-enhanced treatment groups showed improved outcomes compared to synthetic lipids alone, and some formulations improved outcomes to a similar extent as animal-derived surfactant. Robust biophysical mimics of natural surfactant proteins may enable new medical research in ALI treatment.

Biomimetic N-terminal alkylation of peptoid analogues of surfactant protein C.

Surfactant protein C (SP-C) is a hydrophobic lipopeptide that is critical for lung function, in part because it physically catalyzes the formation of surface-associated surfactant reservoirs. Many of SP-C's key biophysical properties derive from its highly stable and hydrophobic α-helix. However, SP-C's posttranslational modification with N-terminal palmitoyl chains also seems to be quite important. We created a new (to our knowledge) class of variants of a synthetic, biomimetic family of peptide mimics (peptoids) that allow us to study the functional effects of biomimetic N-terminal alkylation in vitro. Mimics were designed to emulate the amphipathic patterning, helicity, and hydrophobicity of SP-C, and to include no, one, or two vicinal amide-linked, N-terminal octadecyl chains (providing a reach equivalent to that of natural palmitoyl chains). Pulsating bubble surfactometry and Langmuir-Wilhelmy surface balance studies showed that alkylation improved biomimetic surface activities, yielding lower film compressibility and lower maximum dynamic surface tensions. Atomic force microscopy studies indicated that alkyl chains bind to and retain segregated interfacial surfactant phases at low surface tensions by inducing 3D structural transitions in the monolayer's fluid-like phase, forming surfactant-associated reservoirs. Peptoid-based SP-C mimics are easily produced and purified, and offer much higher chemical and secondary structure stability than polypeptide-based mimics. In surfactant replacements intended for medical use, synthetic SP mimics reduce the odds of pathogen contamination, which may facilitate the wider use of surfactant treatment of respiratory disorders and diseases.

Peptoids: bio-inspired polymers as potential pharmaceuticals.

Peptoids are a developing class of peptide-like oligomers originally invented for drug discovery in the early 1990s. While peptides hold great promise for therapeutic applications, current development of peptide-based pharmaceuticals is hindered by their potential for misfolding and aggregation, and particularly, for rapid in vivo degradation post-administration. Researchers have investigated alternative peptide-like constructs that may be able to circumvent such complications. Peptoids comprise a peptide-based backbone and N-substituted glycines for side chain residues, resulting in complete protease-resistance. Synthesis of peptoid sequences up to 50 units in length allows for controlled sequence composition and incorporation of diverse side chain chemistries. Though the landscape of peptoid structure is not clearly defined, secondary, tertiary, loop, turn, and random structures have been identified. As protease-resistant isomers of peptides, peptoids are being developed as versatile molecular tools in biochemistry and biophysics, and are becoming attractive candidates for therapeutic and diagnostic applications. Peptoids have thus far demonstrated bioactivity as protein mimics and as replacements for small molecule drugs. In this review, we discuss the most recent advances in peptoid research on the therapeutic front in the last few years, including in vitro and in vivo studies in the fields of lung surfactant therapy, antimicrobial agents, diagnostics, and cancer. We particularly focus on the biophysical activity of lipid-associated peptoids and their potential therapeutic applications.

Antimicrobial peptoids are effective against Pseudomonas aeruginosa biofilms.

The resistance of biofilms to conventional antibiotics complicates the treatment of chronic cystic fibrosis (CF). We investigated the effects of peptoids, peptides, and conventional antibiotics on the biomass and cell viability within Pseudomonas aeruginosa biofilms. At their MICs, peptoids 1 and 1-C13(4mer) caused maximum reductions in biomass and cell viability, respectively. These results suggest that peptoids of this class could be worth exploring for the treatment of pulmonary infections occurring in CF patients.

Biophysical mimicry of lung surfactant protein B by random nylon-3 copolymers.

Non-natural oligomers have recently shown promise as functional analogues of lung surfactant proteins B and C (SP-B and SP-C), two helical and amphiphilic proteins that are critical for normal respiration. The generation of non-natural mimics of SP-B and SP-C has previously been restricted to step-by-step, sequence-specific synthesis, which results in discrete oligomers that are intended to manifest specific structural attributes. Here we present an alternative approach to SP-B mimicry that is based on sequence-random copolymers containing cationic and lipophilic subunits. These materials, members of the nylon-3 family, are prepared by ring-opening polymerization of beta-lactams. The best of the nylon-3 polymers display promising in vitro surfactant activities in a mixed lipid film. Pulsating bubble surfactometry data indicate that films containing the most surface-active polymers attain adsorptive and dynamic-cycling properties that surpass those of discrete peptides intended to mimic SP-B. Attachment of an N-terminal octadecanoyl unit to the nylon-3 copolymers, inspired by the post-translational modifications found in SP-C, affords further improvements by reducing the percent surface area compression to reach low minimum surface tension. Cytotoxic effects of the copolymers are diminished relative to that of an SP-B-derived peptide and a peptoid-based mimic. The current study provides evidence that sequence-random copolymers can mimic the in vitro surface-active behavior of lung surfactant proteins in a mixed lipid film. These findings raise the possibility that random copolymers might be useful for developing a lung surfactant replacement, which is an attractive prospect given that such polymers are easier to prepare than are sequence-specific oligomers.

Mimicking SP-C palmitoylation on a peptoid-based SP-B analogue markedly improves surface activity.

Hydrophobic lung surfactant proteins B and C (SP-B and SP-C) are critical for normal respiration in vertebrates, and each comprises specific structural attributes that enable the surface-tension-reducing ability of the lipid-protein mixture in lung surfactant. The difficulty in obtaining pure SP-B and SP-C on a large scale has hindered efforts to develop a non-animal-derived surfactant replacement therapy for respiratory distress. Although peptide-based SP-C mimics exhibit similar activity to the natural protein, helical peptide-based mimics of SP-B benefit from dimeric structures. To determine if in vitro surface activity improvements in a mixed lipid film could be garnered without creating a dimerized structural motif, a helical and cationic peptoid-based SP-B mimic was modified by SP-C-like N-terminus alkylation with octadecylamine. "Hybridized" mono- and dialkylated peptoids significantly decreased the maximum surface tension of the lipid film during cycling on the pulsating bubble surfactometer relative to the unalkylated variant. Peptoids were localized in the fluid phase of giant unilamellar vesicle lipid bilayers, as has been described for SP-B and SP-C. Using Langmuir-Wilhelmy surface balance epifluorescence imaging (FM) and atomic force microscopy (AFM), only lipid-alkylated peptoid films revealed micro- and nanostructures closely resembling films containing SP-B. AFM images of lipid-alkylated peptoid films showed gel condensed-phase domains surrounded by a distinct phase containing "nanosilo" structures believed to enhance re-spreading of submonolayer material. N-terminus alkylation may be a simple, effective method for increasing lipid affinity and surface activity of single-helix SP-B mimics.

Close mimicry of lung surfactant protein B by "clicked" dimers of helical, cationic peptoids.

A family of peptoid dimers developed to mimic SP-B is presented, where two amphipathic, cationic helices are linked by an achiral octameric chain. SP-B is a vital therapeutic protein in lung surfactant replacement therapy, but its large-scale isolation or chemical synthesis is impractical. Enhanced biomimicry of SP-B's disulfide-bonded structure has been previously attempted via disulfide-mediated dimerization of SP-B(1-25) and other peptide mimics, which improved surface activity relative to the monomers. Herein, the effects of disulfide- or "click"-mediated (1,3-dipolar cycloaddition) dimerization, as well as linker chemistry, on the lipid-associated surfactant activity of a peptoid monomer are described. Results revealed that the 'clicked' peptoid dimer enhanced in vitro surface activity in a DPPC:POPG:PA lipid film relative to its disulfide-bonded and monomeric counterparts in both surface balance and pulsating bubble surfactometry studies. On the pulsating bubble surfactometer, the film containing the "clicked" peptoid dimer outperformed all presented peptoid monomers and dimers, and two SP-B derived peptides, attaining an adsorbed surface tension of 22 mN m(-1), and maximum and minimum cycling values of 42 mN m(-1) and near-zero, respectively.

Peptoids that mimic the structure, function, and mechanism of helical antimicrobial peptides.

Antimicrobial peptides (AMPs) and their mimics are emerging as promising antibiotic agents. We present a library of "ampetoids" (antimicrobial peptoid oligomers) with helical structures and biomimetic sequences, several members of which have low-micromolar antimicrobial activities, similar to cationic AMPs like pexiganan. Broad-spectrum activity against six clinically relevant BSL2 pathogens is also shown. This comprehensive structure-activity relationship study, including circular dichroism spectroscopy, minimum inhibitory concentration assays, hemolysis and mammalian cell toxicity studies, and specular x-ray reflectivity measurements shows that the in vitro activities of ampetoids are strikingly similar to those of AMPs themselves, suggesting a strong mechanistic analogy. The ampetoids' antibacterial activity, coupled with their low cytotoxicity against mammalian cells, make them a promising class of antimicrobials for biomedical applications. Peptoids are biostable, with a protease-resistant N-substituted glycine backbone, and their sequences are highly tunable, because an extensive diversity of side chains can be incorporated via facile solid-phase synthesis. Our findings add to the growing evidence that nonnatural foldamers will emerge as an important class of therapeutics.

Lipid composition greatly affects the in vitro surface activity of lung surfactant protein mimics.

A crucial aspect of developing a functional, biomimetic lung surfactant (LS) replacement is the selection of the synthetic lipid mixture and surfactant proteins (SPs) or suitable mimics thereof. Studies elucidating the roles of different lipids and surfactant proteins in natural LS have provided critical information necessary for the development of synthetic LS replacements that offer performance comparable to the natural material. In this study, the in vitro surface-active behaviors of peptide- and peptoid-based mimics of the lung surfactant proteins, SP-B and SP-C, were investigated using three different lipid formulations. The lipid mixtures were chosen from among those commonly used for the testing and characterization of SP mimics--(1) dipalmitoyl phosphatidylcholine:palmitoyloleoyl phosphatidylglycerol 7:3 (w/w) (PCPG), (2) dipalmitoyl phosphatidylcholine:palmitoyloleoyl phosphatidylglycerol:palmitic acid 68:22:9 (w/w) (TL), and (3) dipalmitoyl phosphatidylcholine:palmitoyloleoyl phosphatidylcholine:palmitoyloleoyl phosphatidylglycerol:palmitoyloleoyl phosphatidylethanolamine:palmitoyloleoyl phosphatidylserine:cholesterol 16:10:3:1:3:2 (w/w) (IL). The lipid mixtures and lipid/peptide or lipid/peptoid formulations were characterized in vitro using a Langmuir-Wilhelmy surface balance, fluorescent microscopic imaging of surface film morphology, and a pulsating bubble surfactometer. Results show that the three lipid formulations exhibit significantly different surface-active behaviors, both in the presence and absence of SP mimics, with desirable in vitro biomimetic behaviors being greatest for the TL formulation. Specifically, the TL formulation is able to reach low-surface tensions at physiological temperature as determined by dynamic PBS and LWSB studies, and dynamic PBS studies show this to occur with a minimal amount of compression, similar to natural LS.

Effects of including an N-terminal insertion region and arginine-mimetic side chains in helical peptoid analogues of lung surfactant protein B.

Surfactant protein B (SP-B) is one of two helical, amphipathic proteins critical for the biophysical functioning of lung surfactant (LS) and hence is an important therapeutic protein. This small, complex 79mer has three internal disulfide bonds and homodimerizes via another disulfide bridge. A helical, amphipathic 25mer from the amino terminus (SP-B(1-25)) exhibits surface-active properties similar to those of full-length, synthetic SP-B. In previous work, we created helical, non-natural mimics of SP-B(1-25) based on sequence-specific peptoid 17mers and demonstrated their biomimetic surface activity. Like SP-B(1-25), the peptoids were designed to adopt helical structures with cationic and nonpolar faces. Here, we compare the surface activities of six different helical peptoid analogues of SP-B(1-25) to investigate the importance of mimicking its N-terminal insertion domain as well as its two arginine residues, both thought to be important for the peptide's proper function. Although the peptoid analogues of SP-B(1-25) studied here share many similar features and all functionally mimic SP-B(1-25) to some degree, it is notable that small differences in their sequences and side chain chemistries lead to substantial differences in their observed interactions with a lipid film. A peptoid comprising a hydrophobic, helical insertion region with aromatic side chains shows more biomimetic surface activity than simpler peptoids, and even better activity, by comparison to natural LS, than SP-B(1-25). However, the substitution of lysine-like side chains for arginine-like side chains in the peptoid has little effect on biomimetic surface activity, indicating that interactions of the guanidino groups with lipids may not be critical for the function of these SP-B mimics.